Miniaturization of the bioanalytical process · digestion and application for bioanalysis using...
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Miniaturization of the Bioanalytical Process by Wei Boon, Hon A thesis submitted in fulfilment of the requirements for the degree of Doctor of Philosophy School of Chemistry University of Tasmania Hobart Submitted November 2011
Transcript of Miniaturization of the bioanalytical process · digestion and application for bioanalysis using...
Miniaturization of the Bioanalytical
Process
by
Wei Boon, Hon
A thesis submitted in fulfilment of the requirements for the degree
of
Doctor of Philosophy
School of Chemistry
University of Tasmania
Hobart
Submitted November 2011
ii
DECLARATION
I hereby declare that this thesis contains no material which has been accepted
for the award of any other degree or diploma in any tertiary institution, and to
the best of my knowledge contains no copy or paraphrase of material
previously published or written by any other person, except where due
reference is made in the text of the thesis.
Wei Boon, Hon
This thesis may be made available for loan and limited copying in accordance
with the Copyright Act 1968.
Wei Boon, Hon
University of Tasmania
Hobart
November, 2011
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ACKNOWLEDGEMENTS
This project was supported by a PhD Scholarship from PARC (Pfizer
Analytical Research Center), ACROSS (Australian Center for Research on
Separation Science), together with University of Tasmania. The tuition fee was
covered by a grant from the State Government of Tasmania, Department of
Economic Development and Tourism.
Acknowledgment is due to almost everyone in the University of
Tasmania’s Chemistry department for support and advice throughout the course
of my PhD candidature. In particular, I would like to thanks:
My supervisor, Prof Emily Hilder
For her constructive criticisms and invaluable assistance and guidance throughout my
project research and in the process of producing this thesis. I do believe that I would
never have made it without her endless effort and encouragement.
My co-supervisors, Prof Paul Haddad and Mr Kenneth Saunders
For giving me the opportunity to work with them and for sharing their knowledge,
wisdom and understanding that ultimately has made the following work possible.
All past and present members of ACROSS and PARC
For their friendship, help and collaboration, including Ashraf Ghanem, Anna Nordborg,
Jeremy Deverell, Lea Mauko, Oscar Potter, Mohammed Talebi, Naama Karu, Anthony
Malone, Clodagh Moy, Jonathan Thabano, Greg Dicinoski, Andrew Grosse, Phil Zakaria,
Michael Breadmore, Chris Evenhuis, Marc and Roos Guijt, Eli Marshall, David Schaller,
Esme Candish, Joe Hutchinson, Pavel Nesterenko, Tim Causon and Jessica Gathercole.
Dr Karsten Gömann
For his assistance and sharing his expertise in using the scanning electron microscope in
Central Science Laboratory, University of Tasmania.
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Pfizer staff at Sandwich, UK
The staff members in Pfizer for all the support, friendship, advice and patience during my
research visit in 2010.
The staff and students of the School of Chemistry for providing such a
friendly working environment and made it enjoyable when things weren’t
working in the lab.
Special thanks also go to Dr Ghanem who worked with me for the first 1.5
years and early discussions for necessary directions in synthesizing polymer
monoliths. Also special thanks go to Dr Jeremy Deverell and Dr Jonathan
Thabano for the very helpful comments in the laboratory during the experiments
in progress. Esme Candish and Yi Ting Tan are especially acknowledged for their
kind assistance and meticulous attention to detail for proof-reading my thesis. In
addition, I would also like to thank my parents, family members and those
brothers and sisters from Wellspring Anglican Church Chinese Congregation
who have sculpted me into the person I am today and for providing emotional
support and relaxation over the years. Finally and most importantly, I thank God
for His endless grace and faithfulness:
“But He said to me, ‘My grace is sufficient for you, for My power is made perfect in
weakness.’” (2 Corinthians 12:9).
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LIST OF ABBREVIATIONS
ADME Absorption, distribution, metabolism, and excretion
AIBN ’azoisobutyronitrile
BMA Butylmethacrylate
BVBDMS Bis(p-vinylbenzyl)dimethylsilane
BSA Bovine serum albumin
CEC Capillary electrochromatography
DMN-H6 1,4,4a,5,8,8a-hexahydro-1, 4, 5, 8-exo,endo-
Dimethanonaphthalene
EDMA Ethylene dimethacrylate
ERPA Extended range proteomic analysis
GDMA 1,3-glycerol dimethacrylate
HPLC High performance liquid chromatography
i.d. Internal diameter
IE Ion-exchange
LC-MS/MS Liquid chromatography-tandem mass spectrometry
LMA Lauryl methacrylate
LOD Limit of detection
LOQ Limit of quantification
NBE Norborn-2-ene
PAA Polyacrylamide
RPLC Reversed-phase liquid chromatography
RAFT Reversible addition fragmentation chain transfer
ROMP Ring-opening metathesis polymerization
RSD Relative standard deviations
RAM Restricted access media
SPE Solid-phase extraction
Tris Tris(hydroxymethyl)aminomethane
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LIST OF PUBLICATIONS
Papers in refereed journals and in preparation
1 K. C. Saunders, A.Ghanem, W. B. Hon, E. F. Hilder, and P. R. Haddad,
Separation and sample pre-treatment in bioanalysis using monolithic phases:
A review, Anal. Chim. Acta, 652 (2009) 22. (Chapter 1)
2 W. B. Hon, A. Ghanem, K. C. Saunders, E. F. Hilder, and P. R. Haddad,
Mixed-Mode Porous Polymer Monoliths for Separation of Small Molecule
Pharmaceuticals, in preparation. (Chapter 3)
3 W. B. Hon, K. C. Saunders, E. F. Hilder, and P. R. Haddad, Development of a
trypsin-immobilized monolithic polymer with pipette-tip format for protein
digestion and application for bioanalysis using LC-MS/MS, in preparation.
(Chapters 4 and 5)
Posters and conferences
1. W. B. Hon, A. Ghanem, K. C. Saunders, E. F. Hilder, and P. R. Haddad
Preparation of reversed-phase and cation-exchange mixed-mode porous
sulfopropyl methacrylate polymer-based monolithic capillary for the
separation of pharmaceutical compounds by using capillary-liquid
chromatography, Poster, ASASS (ACROSS Symposium on Advances in
Separation Science), 2008, Tasmania, Australia.
2. W. B. Hon, A. Ghanem, K. C. Saunders, E. F. Hilder, and P. R. Haddad, In situ
preparation of robust porous modified mixed-mode sulfopropyl methacrylate
monolithic capillary for µHPLC analysis of pharmaceutical compounds,
- Poster, 34th International Symposium on High-Performance Liquid Phase
Separations and Related Techniques, 2009, Dresden, Germany. (Poster session
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winner)
- Poster, School of Chemistry Research Evening, 2010, University of Tasmania,
Australia.
3. W. B. Hon, E. F. Hilder, K. C. Saunders, and P. R. Haddad, Development of a
trypsin-immobilized monolithic polymer with pipette-tip format for protein
digestion
- Poster, 35th International Symposium on High-Performance Liquid Phase
Separations and Related Techniques, 2010, Boston, USA.
- Poster, 2nd Separation Science Singapore Conference and Exhibition, 2010,
Singapore. (Top 5 poster)
4. W. B. Hon, E. F. Hilder, K. C. Saunders, and P. R. Haddad, Development of a
trypsin-immobilized monolithic polymer with pipette-tip format for protein
digestion and bioanalysis using LC-MS/MS
- Oral, finalist (second stage) in the Tasmanian Division of AusBiotech-GSK
Student Excellence Awards, 2010, Tasmania, Australia.
- Oral, Sharing Excellent in Research, 2010, University of Tasmania, Australia.
- Oral, RACI Research and Development Topics Conference, 2010, University of
Tasmania, Australia.
- Poster, 26th International Symposium on MicroScale Bioseparations, 2011, San
Diego, USA.
- Poster, Sharing Excellence in Research, 2011, University of Tasmania,
Australia.
- Poster, Chemistry 50th Symposium and Celebrations, 2011, University of
Tasmania, Australia.
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- Oral, 2nd Dalian International Symposium and Exhibition on Chromatography
and Related Techniques, 2011, Dalian, China.
- Poster, 11th Asia-Pacific International Symposium on Microscale Separations
and Analysis, 2011, University of Tasmania, Australia.
Invited lectures
1. P. R. Haddad, W. B. Hon, A. Ghanem, K. C. Saunders, and E. F. Hilder, In situ
Preparation of robust controlled porous modified sulfopropyl methacrylates
polymer-based monolithic capillary for µHPLC analysis, Oral, ISOPS-9 (the
9th International Symposium on Pharmaceutical Sciences), 2009, Ankara, Turkey.
2. P. R. Haddad, E. F. Hilder, D. Schaller, C. Pohl, C. J. Evenhuis, A. Ghanem, W.
B. Hon, and K. C. Saunders, Synthesis and applications of polymer monoliths
for ion-exchange and mixed-mode separations of small molecules, Oral, 8th
Balaton Symposium on High-Performance Separation Methods, 2009 Balaton,
Hungary.
3. P. R. Haddad, E. F. Hilder, D. Schaller, E. Candish, W. B. Hon, K. Saunders, J.
Thabano, M. C. Breadmore, G. Clark, C. Pohl, Ion-exchange stationary phases
based on polymeric monoliths, Oral, Pittcon Conference and Expo, 2011, GA,
USA.
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ABSTRACT
This work presents a systematic study of macroporous polymer monoliths and
their use as stationary phases for microscale separation and as supports for
immobilized trypsin digestion in pipette tip format for bioanalysis.
Reversed-phase/cation-exchange mixed-mode polymer monolithic
columns were prepared in situ within fused-silica capillaries via UV-initiated
free-radical polymerization reaction. Control of the porous properties was
achieved by varying the ratio of porogenic solvents (e.g. 1,4-butanediol and 1-
propanol), the type of porogenic solvents used and the relative amounts of
functional monomers and cross-linker. These columns were successfully used for
the separation of a mixture of selected acidic drugs and -blockers, namely
ketoprofen, ibuprofen, diclofenac, arterenol and propranolol. Monolithic
columns with higher amounts of cross-linker were shown to give better
repeatability. Furthermore, the separation mechanism of the investigated
compounds was shown to be dual mode hydrophobic/ion-exchange interaction
of the analytes with the hydrophobic and cation-exchange monolith. All these
properties demonstrated the potential of these devices for solid-phase extraction
and sample enrichment purposes in miniaturized formats.
Based on the success of the previous work a trypsin-immobilized
monolithic polymer with pipette-tip format was also investigated to explore the
utility of using a tip-based protein digestion methodology in a bioanalytical
setting. The excellent performance of immobilized enzymatic polypropylene
pipette (IMEPP) tips was characterized using MicrOTOF-Q quadrupole time-of-
flight MS and triple quadrupole LC-MS/MS systems. Very high sequence
coverages of over 90% were achieved for the digestion of proteins ranging from
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low molecular weight to high molecular weight proteins with short contact times
prior to MS analysis, which is comparable to 24 h digestion in solution. In
addition, quantitative analysis of target proteins spiked in rat plasma was
demonstrated for the first time with relatively good linearity over a wide range
from 40-1000 ng/mL. The developed IMEPP tips exhibit high plasma loading
capacity up to 40 L of plasma that can be used to yield the highest efficiency for
digestion. The IMEPP tip approach is thus rapid, reliable, and robust suggesting
the potential of this approach to improve sample throughput for pharmaceutical
and pharmacokinetic studies, leading to faster and safer discovery of new drugs
to treat diseases.
The trypsin immobilized polymer monolith was prepared in situ in
syringe-compatible glass tube and evaluated for the digestion of protein.
Preliminary results showed that the enzyme on the immobilized bed exhibited
high proteolytic performance in this special format. The present glass tube
bioreactor provides a promising platform for the full automation, on-line
coupling to detection systems, short sample preparation times and high-
throughput protein digestion.
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TABLE OF CONTENTS
Page
Title ....................................................................................................................................... i
Declaration .......................................................................................................................... ii
Acknowlegdements ............................................................................................................iii
List of Abbreviations ......................................................................................................... v
List of Publications ........................................................................................................... vi
Abstract ............................................................................................................................... ix
Table of Contents ............................................................................................................... xi
Chapter 1: Introduction and Literature Review ....................................... 1
1.1 Introduction ...................................................................................................................1
1.2 Synthesis of organic polymer monoliths for bioanalysis .....................................7
1.3 Monolithic phases for bioanalysis using HPLC ...................................................13
1.4 Monoliths for sample preparation using solid phase extraction .......................20
1.4.1 Off-line sample preparation by SPE ...............................................................22
1.4.2 On-line sample preparation SPE .....................................................................25
1.5 Conclusions and future possibilities ......................................................................27
1.6 Project aims ..................................................................................................................29
1.7 References .....................................................................................................................30
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Chapter 2: Experimental ............................................................................. 37
2.1 Instrumentation ...........................................................................................................37
2.2 Reagents ........................................................................................................................39
2.3 Procedures ....................................................................................................................45
2.3.1 Buffer and standard preparation ....................................................................45
2.3.2 Sample injection ................................................................................................45
2.3.3 Surface modification in fused-silica capillaries ............................................45
2.3.4 Synthesis of polymer monoliths in capillaries ..............................................46
2.3.5 Measurement of ion-exchange capacity ........................................................46
2.3.6 Porosimetry ........................................................................................................47
2.3.7 Surface modification in polypropylene pipette tips ....................................47
2.3.8 Preparation of porous polymer monoliths in PP tips ..................................50
2.3.9 Digestion protocol for immobilized enzyme polypropylene pipette
(IMEPP) tip and MonoTip® Trypsin ..............................................................50
2.3.10 Scanning electron microscopy .......................................................................54
2.4 Calculations ..................................................................................................................54
2.4.1 Permeability based on Darcy’s Law ...............................................................54
2.4.2 Retention factors ................................................................................................54
2.4.3 Sequence coverage ............................................................................................55
2.5 References .....................................................................................................................55
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Chapter 3: Mixed-mode porous polymer monoliths for separation
of small molecule pharmaceuticals .......................................................... 56
3.1 Introduction .................................................................................................................56
3.2 Experimental ................................................................................................................59
3.2.1 Preparation of porous polymer monoliths in fused-silica capillaries .......60
3.2.2 Bulk monolith preparation ..............................................................................60
3.2.3 Standard solutions and sample preparation .................................................63
3.3 Results and discussion ...............................................................................................64
3.3.1 Preparation of polymer monoliths .................................................................64
3.3.2 Characterization of SPMA-based polymer monoliths .................................65
3.3.2.1 Mercury intrusion porosimetry ....................................................................65
3.3.2.2 Scanning electron microscopy (SEM) ..........................................................69
3.3.3 Chromatographic characterization of SPMA monolithic columns ............71
3.3.3.1 Separation mechanism ..................................................................................71
3.3.3.1.1 Reversed-phase interaction ....................................................................71
3.3.3.1.2 Cation-exchange interaction and capacity ............................................73
3.3.3.2 Permeability of SPMA monolithic columns .................................................75
3.3.3.3 Method development and separation performance of SPMA monolithic
columns .....................................................................................................................75
3.3.3.4 Reproducibility of SPMA monolithic columns ............................................79
3.3.4 Effect of cross-linker on the modified SPMA monoliths (SM). ..................81
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3.3.4.1 Combined effects of percentage variations of the cross-linker and porogenic
solvents on modified SPMA monoliths ...................................................................81
3.3.4.2 Mercury intrusion porosimetry of the modified SPMA monoliths ..............84
3.3.4.3 Scanning electron microscopy of the modified SPMA monoliths ................85
3.3.4.4 Separation performance, reproducibility and stability of modified SPMA
monolithic columns ...................................................................................................85
3.4 Conclusions ..................................................................................................................90
3.5 References .....................................................................................................................95
Chapter 4: Development of a Trypsin-Immobilized Monolithic
Polymer with Pipette-Tip Format for Protein Digestion ..................... 98
4.1 Introduction .................................................................................................................98
4.2 Experimental ..............................................................................................................101
4.2.1 Fluorescent assay of protein absorption ......................................................101
4.2.1.1 Photografting of poly(ethylene glycol) methacrylate (PEGMA) ................101
4.2.1.2 Preparing stock solution of protein and fluorescein isothiocyanate (FITC)102
4.2.1.3 Labeling reaction of protein with fluorescein isothiocyanate (FITC-BSA) 102
4.2.1.4 Fluorescent assay of protein adsorption on the pipette tips ........................103
4.2.2 Photografting of 2-vinyl-4,4-dimethylazlactone (VAL).............................104
4.2.3 Immobilization of trypsin on grafted support ............................................104
4.2.4 Sample preparation for protein digestion ...................................................104
4.3 Results and discussion .............................................................................................106
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4.3.1 Surface modification of the polypropylene (PP) tips .................................106
4.3.2 Fluorescence assay of protein adsorption ...................................................111
4.3.3 Enzyme immobilization using azlactone functionalities ..........................113
4.3.4 Effects of trypsin concentration for immobilization on monolithic
support ..............................................................................................................114
4.4 Conclusions ................................................................................................................118
4.5 References ...................................................................................................................119
Chapter 5: Characterization and Application of a Trypsin-
Immobilized Monolithic Polymer with Pipette-Tip Format for
Bioanalysis using LC-MS/MS ................................................................. 121
5.1 Introduction ...............................................................................................................121
5.2 Experimental ..............................................................................................................123
5.2.1 Sample preparation for qualitative analysis ...............................................123
5.2.1.1 Protien digestion in standard buffer ...........................................................123
5.2.1.2 Protein digetion in rat plasma ....................................................................124
5.2.2 Protein identification and multiple reaction monitoring (MRM) design124
5.2.2.1 Sample preparation for MRM development ...............................................124
5.2.2.2 Peptide MRM development ........................................................................125
5.2.3 Characterization of performance of IMEPP tips .........................................127
5.2.3.1 Plasma loading experiment for IMEPP tips ..............................................127
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5.2.3.2 Time coure assay .........................................................................................127
5.2.3.3 Sample clean-up and enrichment functionality assay ................................128
5.2.3.4 Volume test .................................................................................................128
5.2.4 Sample preparation for quantitative analysis in rat plasma .....................128
5.3 Results and discussion .............................................................................................129
5.3.1 Application of IMEPP tips to the digestion of model proteins spiked in
standard buffer and rat plasma .....................................................................129
5.3.2 Design of MRM assays for target proteins ..................................................142
5.3.3 Characterization of the digestion performances of IMEPP tips in Pfizer
UK ......................................................................................................................147
5.3.3.1 Plasma loading capacity of IMEPP tips ....................................................153
5.3.3.2 Time coure study ........................................................................................155
5.3.3.3 Sample clean-up and enrichment functionality assay ................................156
5.3.3.4 Volume test .................................................................................................158
5.3.4 Quantitative analysis of target proteins using LC-MS/MRM method
after digestion using IMEPP tips. ..................................................................160
5.3.5 Operational stability, storage stability and reusability .............................161
5.4 Conclusions ................................................................................................................163
5.5 References ...................................................................................................................163
Chapter 6: Optimization of Permeability and Format of IMEPP Tips
for Proteomic Analysis .............................................................................. 165
xvii
6.1 Introduction ...............................................................................................................165
6.2 Experimental ..............................................................................................................167
6.2.1 Comparison of protein digestion conditions ...............................................167
6.2.2 Protein identification using public protein sequence database ................168
6.2.3 Preparation of highly permeable monoliths ...............................................170
6.2.4 Manipulations of polymer monoliths in situ in polypropylene pipette
tips .....................................................................................................................170
6.3 Results and discussion .............................................................................................170
6.3.1 Optimization of protein digestion conditions.............................................170
6.3.2 Development of a highly permeable IMEPP tip .........................................179
6.3.2.1 Preparation of highly permeable monoliths ................................................179
6.3.2.2 Manipulation of IMEPP tip formats ..........................................................182
6.3.3 Preparation of immobilized enzyme polymer monolith in glass tube for
microdigestion. ................................................................................................196
6.4 Conclusions ................................................................................................................199
6.5 References ...................................................................................................................200
Chapter 7: General conclusions .............................................................. 201
Chapter 1
1
C h a p t e r 1
Introduction & Literature Review
1.1 Introduction
Reversed-phase high performance liquid chromatography (RPLC) has become
one of the most popular analytical techniques over the past 30 years for
quantitative analysis of a wide range of pharmaceutical compounds [1,2]. It has
been used in a variety of fields, including quality control of drugs,
pharmacokinetic studies, and the determination of environmental pollutants or
food additives [1]. The latest paradigms in the process of drug discovery require
smaller quantities of analytes to be separated in an increasingly large number of
samples [3] and in a drug discovery setting, the demand for high quality
bioanalytical assays has grown over recent years. The increase in the number and
potency of new chemical entities and the associated decrease in sample size, have
in turn driven down the limits of quantification required for many bioanalytical
methods. A bioanalytical method now has to be rapid, automated, sensitive,
selective, robust and applicable to large numbers of samples in order to be
considered useful.
Quantitative bioanalysis is an important tool in the process of drug
discovery, especially pertaining to important in vitro and in vivo studies. The data
generated from bioanalytical methods are pivotal in making decisions around
compound progression through the drug discovery process. The need to develop
new bioanalytical methods to support the drug discovery process presents a
continual challenge to the bioanalyst. For example, thousands of samples from in
vitro screens need to be analyzed on a daily basis. Alternatively, quantifying pg/mL
Chapter 1
2
levels of drug-related components in plasma from clinical studies, using a
validated complex bioanalytical method, may be required. More considered
approaches to both the extraction technique and chromatographic separation have
also been successfully adopted for clinical applications.
To further improve the efficiency of the drug discovery process, bioanalysts
have been expected to generate analytical data in a far more efficient manner.
Because of this, the development of more selective extraction techniques and the
optimization of fast chromatographic conditions have come to prominence. Other
advances in analytical chemistry, such as new column chemistries, high-pressure
separations and hyphenated techniques such as liquid chromatography-tandem
mass spectrometry (LC-MS/MS) are common in the development of new
bioanalytical methods. LC-MS/MS is the analytical platform used most commonly
for bioanalysis, as triple quadrupole mass spectrometers offer the required
specificity and sensitivity of detection. LC–MS/MS is ideally suited for
pharmaceutical compounds, owing to the typical compound physicochemical
properties and molecular weight. In addition, LC–MS/MS is specific enough to
allow very rapid chromatographic separations to be performed; it can accept small
volume samples in a micro-titre plate format, and it can also be used to monitor
multiple analytes in the same complex sample (such as a plasma extract). LC-
MS/MS has become the most widely used analytical platform for quantifying
drugs and their metabolites at low concentration levels in biological matrices [4].
For rapid LC–MS/MS bioanalysis, most laboratories use short analytical columns
(3–10 cm), with relatively high flow-rates (e.g. 1 mL/min), small particles (5 μm or
less) and fast gradients (10% -90% organic modifier) to achieve full analysis in less
than 5 min per sample [5]. Single ion monitoring MS and multiple reaction
monitoring MS/MS techniques provide great detection power. Since its
Chapter 1
3
establishment in the early 1990s, LC-MS/MS has revolutionized the development
of assays for new therapeutic agents in biomatrices [4,6,7], replacing more
traditional high performance liquid chromatography (HPLC) detection techniques,
such as ultra-violet or fluorescence.
The development of new analytical methodologies for high-throughput
bioanalysis has focused on the better utilization of the significant capital
investment made in LC-MS/MS instrumentation and automation. Advances in on-
line and off-line sample preparation (e.g. sorbent chemistry, micro-plate formats,
turbulent flow chromatography and column switching) and further advances in
HPLC technology, which lead to enhancements in speed, sensitivity, and
resolution (e.g. ultra-high pressure, capillary- and nano- HPLC ) [8-17], have also
been implemented.
Sample preparation is a key step in quantitative bioanalysis and can often be
a bottleneck in the process of developing a robust and efficient bioanalytical
methodology [4,18]. The sample preparation stage is often the most labor-
intensive, time-consuming and error-prone process in a bioanalytical method [4].
This can be attributed to the complex nature of macromolecular compounds, such
as proteins and non-volatile endogenous substances, that have to be removed from
a biological sample and separated from the analytes to eliminate matrix
interferences prior to the LC-MS/MS analyses [19]. Sample preparation can also
serve to pre-concentrate the sample while removing the endogenous matrix
interferences at the same time. This leads to an increase in sensitivity and an ability
to subsequently use faster, less well resolved chromatographic separations. Sample
preparation is typically achieved by liquid-liquid extraction [20], solid-phase
extraction (SPE) [13,21], protein precipitation [22], and on-line methods with HPLC
[23]. In addition, as the number of samples increases, there has been a trend to
Chapter 1
4
develop different formats of sample preparation devices, more selective sorbents
for sample clean-up and enrichment, as well as fully automated analytical
techniques [24,25]. The introduction of automated sample pre-treatment
approaches has greatly increased the efficiency of the bioanalysis process [26].
Automated SPE in a 96-well plate format is now an established technique for
sample clean up for biological samples [10].
One way to overcome some of the challenges associated with using fast
LC/MS/MS is the use of monolithic phases. These have a number of potential
advantages over more conventional silica-based particulate materials, as outlined
in Table 1.1. Polymer monoliths can be adapted to most bioanalytical situations,
such as larger scale sample preparation and small-scale capillary- or nano- HPLC
separations, and therefore their use for bioanalysis has increased over recent years.
These materials have some interesting properties when compared with the more
traditional particulate materials, such as their high permeability for liquids and
biological samples, thus making them ideal for SPE. The relative ease of synthesis,
high control of shape, porosity and selectivity give the bioanalyst many options in
the choice of bioanalytical applications and formats for monolithic phases.
Additionally, the option of operating at high or low flow-rates, stability at high pH
as compared to similar silica-based materials and low column pressure drop, can
yield flexibility and speed for HPLC separations. These phases can be prepared in
situ within narrow capillaries and applied to micro-scale separations and even
further miniaturized for micro-fabricated applications. In summary, monolithic
phases can be used in a variety of formats and can be prepared in a range of
formats including HPLC columns, SPE plates, pipette tips or even synthesized in
narrow capillaries for nano-flow chromatography.
Organic polymeric monoliths can take two structurally different forms: (i)
Chapter 1
5
Table 1.1: Advantages, disadvantages of using monoliths as compared to
alternative phases for bioanalytical applications.
Monolithic phases
Other phases
(i.e. packed particles)
Advantages Low cost, relative ease of in situ preparation (e.g.
thermal initiation, redox reagents, UV and
gamma radiation).
Mechanically robust, no void volumes formed
with conventional LC flow-rates.
Can control the porous properties by varying the
compositions of monomers and porogens for the
separations of large and small-molecules.
High hydraulic permeability and the dominance
of the convection over the diffusion mechanism
of mass-exchange under dynamic conditions that
allow the separation to be carried out at
extremely high flow-rates.
Flexible synthesis means that monoliths can be
modified with almost any functionality,
including ion-exchange, affinity, chiral, mixed-
mode, restricted access, hydrophobic and
hydrophilic to tailor the stationary phases for
different analytes.
Can be molded into any shape (capillary,
column, micropipette tips, microfluidic channel
on a chip).
Relatively biocompatible, due to open porous
structure. Could be used for SPE column for over
200 times for the analysis of human plasma
without loss of efficiency.
Can withstand more extreme conditions (i.e. pH
working range from 2 to10).
High surface area.
Higher column
efficiencies.
Small particle sizes
and high operating
pressures.
Diverse column
chemistries and
HPLC column
dimensions.
Validated
applications/assays.
Chapter 1
6
Disadvantages Lower surface area, lower binding capacity.
Lower column efficiencies and HPLC column-to-
column repeatability.
Narrow column chemistries and column
dimensions commercially available.
Limited use in routine analysis due to the
limitation of commercial suppliers.
Higher backpressure
(slow diffusional mass
transfer).
For extraction, sorption
materials, such as
beads requires extra
instruments to generate
high-pressure for the
sample solution to pass
through the media. The
intrinsic problem of all
particulate media is
their inability to
completely fill the
available space. The
channeling between
particles reduces
extraction efficiencies
and can adversely
affect flow
characteristics.
Fabrication of packed
capillary columns
requires a tremendous
amount of skill because
of small i.d. particles
used.
Need for frits,
undesired interactions
of the frits with some
analytes can make the
utilization of packed
capillary columns very
problematic.
Narrow pH stability
range.
Chapter 1
7
homogeneous gels and (ii) rigid porous polymers. Homogeneous gels can be
applied as visually transparent or opalescent separation media and in principle
they represent an almost ideal chromatographic support because of their high
porosity and the fact that the eddy diffusion is negligible. An example of a
homogeneous gel is the low cross-linked polyacrylamide (PAA), which is a soft
and highly swollen material. On the other hand, rigid porous polymeric monoliths
consist of a single piece of highly cross-linked macroporous polymer with
interconnected pores and greater structural rigidity separations with low column
backpressure and fast mass transfer kinetics [1-3].
Recently, Guiochon [27], Smith and Jiang [28] and Potter and Hilder [29]
have reviewed some general aspects of monolithic materials and their applications.
In this chapter the focus is on recent developments in the use of monoliths (with
particular emphasis on polymer monoliths) in the applied area of bioanalysis. The
synthesis of polymer monoliths is discussed, followed by the use of monoliths for
bioanalysis by HPLC and for SPE. Finally, some future directions are indicated.
1.2 Synthesis of organic polymer monoliths for bioanalysis
Despite the widespread progress in monolith synthesis, the preparation of
monoliths may often remain a trial-and-error procedure. Furthermore, scale-up of
the synthesis to larger diameter formats above typical analytical dimensions
remains a challenge due to the high exothermic level of the polymerization
reaction which becomes paramount in the case of large-volume monoliths. This can
result in significant radial temperature gradients, leading to a non-uniform pore
structure. Danquah and Forde [30] employed a novel synthetic technique using a
heat expulsion mechanism to prepare a large methacrylate monolith (40 mL) with a
homogeneous radial pore structure along its thickness. This large monolith was
Chapter 1
8
used for the rapid purification of pDNA from clarified bacteria lysate using
elevated flow-rates under moderate pressure drops. The same authors reported the
preparation of macroporous methacrylate monolithic material with controlled pore
structure using an unstirred mould through precise control of the polymerization
kinetics and parameters. The results revealed that the control of the kinetics of the
overall process through changes in reaction time, temperature and overall
composition, such as cross-linker and initiator contents, allowed the fine tuning of
the macroporous structure [31]. In contrast to this work, Chen et al. [32] directly
prepared a supermacroporous monolithic cryogel by in situ cryo-copolymerization
in a stainless steel cartridge using methacrylic acid as functional monomer and
polyethylene glycol diacrylate as cross-linker. The resulting highly cross-linked
(90%, molar ratio) cryogel had more uniform super macropores as compared to the
poly(acrylamide)-based cryogels. The viability of this cryogel as a medium was
demonstrated through the separations of lysozyme from chicken egg white and
water-soluble nanoparticles from crude reaction solution.
A weak ion-exchange grafted methacrylate monolith was prepared by
Frankovic et al. [3] by grafting the methacrylate monolith with glycidyl
methacrylate and subsequently modifying the epoxy groups with diethylamine. A
comparison of the binding capacity for the non-grafted and grafted monolith was
performed using β-lactoglobulin, bovine serum albumin (BSA), thyroglobulin, and
plasmid DNA (pDNA). The results revealed that the grafted monolith exhibited 2
to 3.5-fold higher capacities (as compared to non-grafted monoliths). Furthermore,
the maximum pDNA binding capacity was reached using 0.1 M NaCl in the
loading buffer and no degradation of the supercoiled pDNA form was detected.
The grafted monolith exhibited lower efficiency than the non-grafted version,
Chapter 1
9
however baseline separation of pDNA from RNA and other impurities was
achieved from a real sample.
Monolithic materials have been prepared for small drug molecule
bioanalysis ranging from high-throughput separations in combinatorial chemistry
through to validated clinical assays. For example, Aoki et al. have described a new
approach to prepare polymer monoliths with morphology tailored for HPLC
application to small solutes such as drug candidates [33]. Polymer monoliths based
on 1,3-glycerol dimethacrylate (GDMA) were prepared by in situ photo-initiated
free radical polymerization (UV-irradiation at 365 nm). The photo-polymerization
was carried out with a mono-disperse ultra-high molecular weight polystyrene
solution in chlorobenzene uniquely formulated as a porogen. The poly-GDMA
monoliths were prepared in bulk, rod and capillary formats and these showed a
bicontinuous network-like structure featured by their fine skeletal thickness
approaching sub-m size. This monolithic structure was considered to be a time-
evolved morphology frozen by UV-irradiation via visco-elastic phase separation
induced by the porogenic polystyrene solution. The UV-initiated poly-GDMA
capillary monolith demonstrated a sharp elution profile affording higher column
efficiency and permeability as compared to the thermally prepared capillary of the
same pore size. This investigation showed that poly-GDMA monoliths with a well-
defined bicontinuous structure could be prepared reproducibly by photo-initiated
radical polymerization via visco-elastic phase separation using the unique porogen.
Roohi et al. [34] reported an interesting alternative to reversed-phase (RP)
monolithic columns using a convenient coupling route of a thermo-responsive
polymer to hydrophilic silica monoliths. Poly(N-isopropylacrylamide) was
polymerized in solution via a reversible addition fragmentation chain transfer
(RAFT) polymerization technique and coupled then in situ onto an amino-modified
Chapter 1
10
silica monolithic column. These columns were compared with reversed-phase C18
monolithic columns in the separation of steroids under isocratic conditions using
an aqueous mobile phase. The separation was optimized by changing the
temperature rather than by changing the mobile phase composition. Another rapid
method for profiling steroids with a wide range of polarity has been developed by
Colosi et al. [35]. Utilizing HPLC equipped with MS detection and a monolithic LC
column, steroids were detected and quantified using testosterone-d3 as the internal
standard. The method was compared to two similar methods using a traditional
particulate column in terms of number of steroids eluted, peak area repeatability,
limits of detection and overall analysis time. The monolithic method eluted the
steroids in a 20 min analysis time, whereas the particulate methods required up to
45 min [35].
Levkin et al. [36] prepared poly(lauryl methacrylate-co-ethylene glycol
dimethacrylate) and poly(styrene-co-divinylbenzene) stationary phases in
monolithic format using thermally initiated free radical polymerization within
polyimide chips featuring channels having a cross-section of 200 m 200 m and
a length of 6.8 cm. These chips were then used for the separation of a mixture of
proteins including RNase A, myoglobin, cytochrome, and ovalbumin, as well as
peptides. Both the monolithic phases based on methacrylate and on styrene
chemistries enabled the rapid baseline separation of most of the test mixtures. The
best performance was achieved with the styrenic monolith leading to fast baseline
separation of all four proteins in less than 2.5 min. The in situ monolith preparation
process afforded microfluidic devices exhibiting good batch-to-batch and injection-
to-injection repeatability [36]. Furthermore, Zhao et al. [37] developed a capillary
chromatographic technique for the separation and detection of proteins, taking
advantage of the specific affinity of aptamers and the porous property of the
Chapter 1
11
monolith. A biotinylated DNA aptamer targeting cytochrome c was successfully
immobilized on a streptavidin-modified polymer monolithic capillary column. The
aptamer, having a G-quartet structure, could bind to both cytochrome c and
thrombin, enabling the separation of these proteins from each other and from the
unretained proteins. Elution of strongly bound proteins was achieved by
increasing the ionic strength of the mobile phase. The following proteins were
tested using the aptamer affinity monolithic columns: human IgG, Hb, transferrin,
human serum albumin, cytochrome c, and thrombin. The benefit of porous
properties of the affinity monolithic column was demonstrated by the selective
capture and pre-concentration of thrombin at low ionic strength and the
subsequent rapid elution at high ionic strength. The combination of the polymer
monolithic column and the aptamer affinities makes the aptamer-modified
monolithic columns useful for protein detection and separation.
Sinner et al. [38] prepared monolithic capillary columns via ring-opening
metathesis polymerization (ROMP) using norborn-2-ene (NBE) and 1,4,4a,5,8,8a-
hexahydro-1, 4, 5, 8-exo,endo-dimethanonaphthalene (DMN-H6) as monomers. The
monolithic polymer was copolymerized with Grubbs-type initiator
RuCl2(PCy3)2(CHPh) and a suitable porogenic system within the confines of fused
silica capillaries of different inner diameters (i.d.). Capillary monoliths of 200 m
i.d. showed good performance in terms of retention times, with relative standard
deviations (RSD) of 1.9% for proteins and 2.2% for peptides. However, the
separately synthesized capillary monoliths revealed pronounced variation in back
pressure with RSD values of up to 31%. These variations were considerably
reduced by the cooling of the capillaries during polymerization. Using this
optimized preparation procedure capillary monoliths of 100 and 50 m i.d. were
synthesized and the effects of scaling down the column i.d. on the morphology and
Chapter 1
12
on the repeatability of the polymerization process were investigated. The
applicability of ROMP-derived capillary monoliths to a separation problem
common in medical research was assessed. A 200 m i.d. monolithic column
demonstrated excellent separation behavior for insulin and various insulin
analogs, showing equivalent separation performance to Vydac C4 and Zorbax C3-
based stationary phases. Moreover, the high permeability of monoliths enabled
chromatographic separations at higher flow-rates, leading to shortened analysis
times. For the analysis of insulin in human biofluid samples, enhanced sensitivity
was achieved using a 50 µm i.d. ROMP-derived monolith [38].
Wieder et al. [39] prepared hydrophobic organosilane-based monolithic
capillary columns by thermally initiated free radical polymerization within the
confines of 200 µm i.d. fused silica capillaries. A novel cross-linker, bis(p-
vinylbenzyl)dimethylsilane (BVBDMS), was copolymerized with p-methylstyrene
(MS) in the presence of 2-propanol and toluene, using ’azoisobutyronitrile
(AIBN) as initiator. Monolithic capillary columns, differing in the total monomer,
microporogen content and nature (2-propanol versus toluene, THF or CH2Cl2)
were fabricated and the chromatographic efficiency of each monolith for the
separation of proteins, peptides and oligonucleotides, was evaluated. The pressure
drop versus flow-rate measurements showed the prepared poly(p-methylstyrene-
co- bis(p-vinylbenzyl)dimethylsilane) (MS/BVBDMS) monoliths to be mechanically
stable and swelling propensity factors of 0.78–1.10 indicated high cross-linking
homogeneity [39].
For the separation of peptides with gradient-elution liquid chromatography,
Pruim et al. [1] prepared a poly(butyl methacrylate-co-ethylene glycol
dimethacrylate) (BMA-co-EDMA) monolithic capillary column. The conditional
peak capacity was used as a metric for the performance of this column, which was
Chapter 1
13
compared with a capillary column packed with C18-modified silica particles. The
retention of the peptides was found to be less on the BMA-co-EDMA column than
on the particulate C18 column. To obtain the same retention in isocratic elution an
acetonitrile concentration approximately 15% (v/v) lower was used in the mobile
phase. The retention window in gradient elution was correspondingly smaller with
the BMA-co-EDMA column. The relationship between peak width and retention
under gradient conditions was studied in detail. It was found that in shallow
gradients, with gradient times of 30 min and more, the peak widths of the least
retained compounds strongly increased with the BMA-co-EDMA column. This was
attributed to the fact that these compounds were eluted with an unfavorably high
retention factor. With shallow gradients the peak capacity of the BMA-co-EDMA
column (≈90) was clearly lower than that of a conventional packed column (≈150).
On the other hand, with steep gradients, when the components were eluted with a
low effective retention factor, the performance of the BMA-co-EDMA column was
relatively good. With a gradient time of 15 min similar peak widths and thus
similar peak capacities (≈75) were found for the packed and the monolithic
columns. Two strategies were investigated to obtain higher peak capacities with
methacrylate monolithic columns. The use of lauryl methacrylate (LMA) instead of
BMA gave an increase in retention and narrower peaks for early eluted peptides.
The peak capacity of the LMA column was ≈125 in a 60 min gradient. Another
approach used a longer BMA-co-EDMA column which resulted in a peak capacity
of ≈135 in 60 min.
1.3 Monolithic phases for bioanalysis using HPLC
The wide variety of applications of silica monolithic phases for HPLC has been
summarized by Unger et al. [10], including high-throughput analysis of drugs and
Chapter 1
14
metabolites [11-15,40], separation of complex biological samples (e.g. biofluid
samples, proteins and peptides) [16,17,19-21,24,41] and separation of biological
samples in more complex multi-dimensional HPLC [22,23,25,42]. High-throughput
bioanalysis in a drug discovery setting has promoted the change from simple
isocratic elution towards relatively higher flow-rates and fast gradient elution. Fast
generic methodology with ballistic gradients, high flow-rates and shorter columns
(typically of length 10-50 mm and internal diameters 2.1-4.6 mm) packed with 3-5
m silica-based phases were used in early applications of high-throughput LC-
MS/MS assays to dramatically reduce the chromatographic run times in
bioanalysis and ADME (absorption, distribution, metabolism, and excretion)
screening [43,44]. Ultra-fast, high-pressure chromatographic LC-MS/MS
approaches with short columns and sub-2 µm particle sizes have been reported for
quantitative bioanalysis [45-47]. The corresponding increase in column back
pressure and smaller sample injection volumes using this approach with biological
samples can be avoided using flow-porous monolithic columns. Separation using
an eluent flow-rate gradient can be performed on monolithic columns due to their
structure and unique hydrodynamic characteristics [48]. The use of monolithic
silica columns for screening applications and discovery bioanalysis by fast gradient
LC-MS/MS, is now well established [49-52].
Monolithic silica and polymer columns have been commercialized by
Merck (Darmstadt, Germany) and by Dionex (Sunnyvale, USA) under the brand
names of ChromolithTM (silica) and ProSwiftTM respectively. Several reviews have
also described the use of a gradient approach for ultra-fast bioanalysis, with run
times ranging from less than 10 min for certain separations down to 30 s or less for
more high-throughput applications [53-55]. Tzanavaras and Themelis [56]
performed high-throughput assay of acyclovir and its major impurity guanine
Chapter 1
15
using the ChromolithTM (100 mm x 4.6mm i.d., Merck) column and a flow-gradient
approach in HPLC system [56]. Papp et al. [52] presented a rapid and sensitive
method for quantitation of montelukast in sheep plasma using LC-MS/MS. The
method used a Chromolith RP column (25 mm x 4.6 mm i.d., Merck) with the
gradient concentration of acetonitrile (30-95% acetonitrile in 1 min, 95% for the
next 0.2 min and re-equilibration to 30% over 0.3 min) and a simple one-step
protein precipitation sample preparation step, which has a limit of quantification of
0.36 ng/mL. A total run time of 1.5 min was achieved with the precision below 5%
and an overall relative standard deviation of 8%. In summary, the use of short
monolithic columns with fast gradient approaches parallels the increased
prominence of bioanalysis and sample numbers to be analyzed in a drug discovery
setting.
Monolithic HPLC has been successfully implemented to improve the
efficiency and resolution for bioanalytical HPLC assays. In particular, monolithic
phase HPLC separations have been established for fast reversed-phase gradient
HPLC, with an initial divert to waste period allowing for more selective separation
and reduction of ion suppression phenomena with MS detection. A simplified
work-flow for this type of assay is indicated in Figure 1-1. This very fast gradient
HPLC system allows direct analysis of biological samples that have undergone
minimal sample pre-treatment (e.g. protein precipitation using acetonitrile). The
initial divert to waste period reduces the introduction of polar components, non-
volatile salts, soluble proteins and other endogenous material to the MS.
Employment of a fast gradient achieves some separation of the analytes from the
remaining endogenous material and gives sharper eluting peaks. Therefore, these
fast gradient systems generally exhibit reduced ionization suppression effects as
compared to those previously employed using isocratic HPLC conditions (with a
Chapter 1
16
Automated
sample prep,
dilution, QCs,
calibration curve
LC-MS/MS
quantification
Target
drug molecule
In plasma
Addition of
analogue IS
Automated
sample prep,
dilution, QCs,
calibration curve
Automated
sample prep,
dilution, QCs,
calibration curve
Automated
sample prep,
dilution, QCs,
calibration curve
LC-MS/MS
quantification
Target
drug molecule
In plasma
Target
drug molecule
In plasma
Addition of
analogue IS
Addition of
analogue IS
Addition of
analogue IS
Figure 1-1: Typical assay work-flow for a small molecule drug in plasma in an on-line configuration using a monolithic
HPLC column.
Chapter 1
17
high percentage organic mobile phase composition) [52]. The fast gradient systems
are simple to set up, requiring only a binary HPLC pump and a single switching
valve. A schematic diagram for this type of set-up is indicated in Figure 1-2.
Miniaturization of these HPLC column separations has recently been developed,
giving MS sensitivity as well as solvent and sample consumption advantages, and
narrow-bore and capillary columns are being employed routinely in bioanalytical
laboratories.
Miniaturization based on decreasing the i.d. of the chromatographic column
has driven the development of HPLC column formats from narrow-bore (2.1 mm <
i.d. < 4 mm) to nano-bore (25 m < i.d. < 100 m) or even smaller columns (open
tubular liquid chromatography < 25 m) for nano analysis of biological molecules.
Four factors have promoted the trend to develop smaller LC columns. The recent
developments in capillary-LC have been aimed to enhance the separation
efficiency characteristics of capillary techniques, particularly where many analytes
need to be separated. The second factor is the overwhelming growth of LC-MS/MS
in drug discovery and development; this has in turn spurred the need to interface
the MS source more optimally with the column flow-rates to yield more efficient
ionization and sensitivity. The third factor is the requirement of capillary-LC
columns to analyze smaller-sized bioanalytical samples with less mobile phase
consumption. Finally, the development of the chromatographic hardware, in
particular, pumps capable of reproducibly delivering flow-rates in the nL/min, has
greatly enhanced the practical implementation of miniaturized systems.
Monolithic capillary columns were initially developed for capillary
electrochromatography (CEC) and stimulated the introduction of this format for
HPLC separations [57]. Capillary-HPLC monolithic columns provide an alternative
approach to conventional phases in the analyses of protein molecules, because of
Chapter 1
18
Figure 1-2: Diagram showing the divert valve set-up for a monolithic gradient
HPLC system, for direct injection of biofluids (e.g. protein precipitated plasma
or diluted urine). Position A is held in high aqueous conditions with the HPLC
eluent being directed to waste, thereby removing polar interferences and non-
volatile salts. After this hold period the valve is switched to position B to direct
the eluent into the mass spectrometer and a fast gradient is performed to elute
the analytes. This process is fully automated and is able to eliminate the need
for a separate SPE procedure.
Chapter 1
19
one-step fabrication process and faster analysis times [58]. Most of the publications to
date have focused on the synthesis and characterization of monolithic materials for
capillary- or nano-LC with internal diameters of approximately 25-320 m [58-
64]. Urban and Jandera [26] reviewed the syntheticpolymethacrylate-based
monoliths in capillary-LC and their applications. Numerous highly important
applications of micro-LC are in the field of analyses of low- and high-molecular
weight biologically active solutes, typically for protein and peptides [64-66].
Zhang et al. [67] recently developed a LC-MS platform, known as the extended
range proteomic analysis (ERPA) for comprehensive protein characterization at
ultra-trace level. Large peptides with or without post-translational modifications
were finely separated with high resolution using nano-bore (20 and 50 m i.d.)
polystyrene divinylbenzene (PS-DVB) monolithic columns. High sequence
coverage (>95%) analysis for -casein and epidermal growth factor receptor at
low-molecular levels were achieved using the nano-bore polymer monolithic
column. This methodology has immense potential for the characterization of
biologically important proteins at trace level and the facilitation of biomarker
discovery [61]. The living functionalities of monoliths that allow grafting of
surfaces with functional polymer chains open up a new approach to capillary
column technologies and chemistries [57]. However, there are relatively few
publications that focus on the synthesis and fabrication of polymer monoliths for
the bioanalysis of small molecules using nano-bore HPLC columns.
Most monolithic columns are shorter in lengths, so as to achieve faster
separation and to ensure the consistency of the monolithic morphology throughout
the column. Hosoya et al. [68] prepared novel wired chip devices for -HPLC
analyses where the monolithic capillary column is 95 cm length. The latter was
prepared using a tri-functional epoxy monomer, tris(2,3-epoxypropyl)isocyanurate
Chapter 1
20
with a diamine, 4-[(4-aminocyclohexyl) methyl] cyclohexylamine. The prepared
column was evaluated by observing the sectional structure of the column with a
scanning electron microscope and also by -HPLC. The repeatability in the
preparation of the long capillary columns was extensively examined for
applications of novel wired chip devices. Furthermore, the wired chip device
column showed that its high performance was maintained even after chip
preparation [68].
1.4 Monoliths for sample preparation using solid phase extraction
Solid phase extraction (SPE) has become one of the most popular sample
preparation strategies for the extraction and pre-concentration of small-molecule
drugs in biological samples [69,70]. SPE can offer the benefits of a selective clean-
up, lower detection limits, high accuracy and precision, amenability to
automation and also extended column lifetimes [71]. SPE also offers a large
choice of sorbent phases and formats, allowing flexibility with respect to assay
development, miniaturization and sample throughput.
SPE sorbents are commonly based on chemically bonded phases of silica,
cross-linked polymers or graphitized carbon, with bonded silica-based materials
especially dominating the field [72,73]. Particles of the SPE phase can be packed in
a cartridge, micro-plate or column [73]. However, there are some inherent
limitations of silica-based materials, which include the presence of polar silanol
groups, low surface area and narrow pH stability range. Furthermore, an intrinsic
problem of all particulate media is their inability to completely fill the available
space, with flow-channeling between particles leading to reduced extraction
efficiencies and adverse flow characteristics [71,74]. This has led to the
development of alternative SPE formats, such as disk type materials with
Chapter 1
21
embedded small sorbent particles [74] or micro-sized disks with bonded phases
[75]. By using the disk approach, better extraction recovery can be obtained due to
the superior flow control between samples [71,74,76]. The extraction disks used for
SPE are normally prepared by punching from larger sheets of material, but the
limitation of this approach is that it often results in low sample loading capacity
[77].
In situ preparation of SPE materials with larger surface area and capacity
can be easily achieved with the use of monolithic materials [15,74]. The unique
properties of monoliths, in particular their high permeability for liquid biological
samples, operation at high flow-rates without loss of efficiency [78,79], low cost,
pH stability and ease of preparation, as well as adjustable shape, porosity and
selectivity, allow them to meet the requirements of modern miniaturized SPE
materials designed specifically for automated operation [7,71].
Many different options exist for the off-line and on-line coupling of SPE to
HPLC. The most common approach is the off-line configuration, although it can be
a time-consuming process to set up this methodology in an automated fashion,
unless specific robotic interfaces are used. In addition, technical difficulties may
arise when dealing with smaller sample volumes, and pre-concentration methods
and complex elution protocols are also often required. In on-line approaches [80-
82], the sample preparation step is embedded into the chromatographic system
and manual intervention is minimized, thereby increasing efficiency and sample
throughput [83]. Both silica- and polymer-based monolithic sorbents have
significant advantages over packed particles for SPE and hence monolithic phases
are becoming more popular as sorbent materials for SPE. Several reviews have
addressed the applications of silica- and polymer-based monoliths for bioanalytical
sample preparation [27,45,74,83].
Chapter 1
22
1.4.1 Off-line sample preparation by SPE
Off-line SPE can be optimized independently from the ensuing HPLC separation
and can be easily automated [84]. For this reason, off-line SPE has been
frequently performed using a range of commercial disposable cartridges or disks
[69,85]. SPE techniques in 96-well format are widely adopted in automated
higher throughput quantitative bioanalysis and hence are also well-suited for
LC-MS/MS applications [85,86]. The associated liquid transfer steps, including
preparation of calibration standards and quality control samples as well as the
addition of the internal standard are usually performed using robotic liquid
handling systems [83,87]. The principles and application details of automated 96-
well SPE have been documented in a number of publications [88,89], and a
simplified work-flow for this type of assay is shown in Figure 1-3. In addition,
several related small volume 96- and 384-well SPE devices have been described
and these provide the benefits of lower solvent requirements, minimal
desorption volumes and decreased void volume [84,90-92]. Combinations of a
number of techniques (e.g. protein precipitation followed by SPE) and more
intricate extraction protocols have also been developed to achieve high purity of
the sample extract as well as high sample throughput [93].
Monolithic materials have recently been introduced in the field of SPE and
have potentially great impact in some bioanalytical applications [82]. SPE using
monolithic silica gel cast into a pipette tip was reported by Miyazaki et al. [94] for
the purification of biological samples. The surface of monolithic silica can be
chemically modified readily to provide selective extraction of analytes with
specific characteristics (e.g. hydrophilic, hydrophobic, phosphorylated form, etc.)
Polymer monoliths in SPE can be prepared in situ in a wide range of formats,
including capillary, micropipette tips, and microfluidic channels and can offer
Chapter 1
23
Automated
96-well SPE
Automated
sample prep,
dilution, QCs,
calibration curve
LC-MS/MS
quantification
Target
drug molecule
in plasma
Sample
evaporation/
reconstitution
Addition of stable
labelled IS
Automated
96-well SPE
Automated
96-well SPE
Automated
96-well SPE
Automated
sample prep,
dilution, QCs,
calibration curve
Automated
sample prep,
dilution, QCs,
calibration curve
Automated
sample prep,
dilution, QCs,
calibration curve
LC-MS/MS
quantification
Target
drug molecule
in plasma
Target
drug molecule
in plasma
Sample
evaporation/
reconstitution
Sample
evaporation/
reconstitution
Sample
evaporation/
reconstitution
Addition of stable
labelled IS
Addition of stable
labelled IS
Addition of stable
labelled IS
Figure 1-3: Typical assay work-flow for a small molecule drug in plasma in an off-line configuration with automated
SPE.
Chapter 1
24
exciting possibilities for micro-SPE [18,29,84,94]. Using this approach a wide
range of surface functionalities can be accessed either through co-polymerization
of suitable functional monomers, simple direct chemical modification of the base
polymer or by photografting [95] or coating a suitable monolithic scaffold with
functionalized nanoparticles [96,97]. The approach of coating with nanoparticles
is particularly attractive as suitable nanoparticles can be synthesized with almost
any functionality, including ion-exchange, affinity, chiral, mixed-mode, restricted
access, hydrophobic and hydrophilic, and the same scaffold can be used for a
wide range of nanoparticles with different chemistries.
There are numerous reports describing off-line SPE using polymer
monoliths, with subsequent coupling to HPLC for bioanalysis [98-101]. Altun et
al. and Abdel-Rehim et al. [18,102-104] introduced a sample preparation
technique using a set of polypropylene tips containing a plug of an in situ
polymerized methacrylate-based monolithic sorbent for use with 96-well plates.
These sorbents have been used successfully for the extraction and quantification
of -blockers [104] and anaesthetics [103] from human plasma by LC-MS/MS.
This novel design permitted 96 samples be extracted in approximately 2 min,
with only microlitre volumes of solvent being required for elution. In addition,
the unique properties of monolithic materials facilitate a low pressure drop, thus,
reducing the risk of blockages due to plasma samples even at higher flow rates.
Moreover, they also provide high analyte binding efficiency and yield good
recoveries as a result.
This approach can be further extended to include immobilized enzymes
for sample digestion. Ota et al. [105] described a novel silica monolith within a
pipette tip, where trypsin was successfully immobilized via an aminopropyl
group. The trypsin-immobilized monolith was used for rapid digestion of
Chapter 1
25
reduced and alkylated proteins with only minimal manual operations prior to
chromatographic analysis, and was evaluated for high-throughput trypsin
proteolysis of bio-substances in proteomics [105].
1.4.2 On-line sample preparation SPE
In an HPLC system for bioanalytical applications, monolithic columns can serve
either as an on-line extraction cartridge or analytical column, or combinations of
this in a column switching or multi-dimensional set-up. Performing the SPE
process as part of the HPLC system has recently gained popularity as an
automated bioanalytical technique. The major advantage of this approach is that
it can incorporate the sample preparation, analyte enrichment, HPLC separation
and MS detection steps into a single system. In-line SPE is based on direct
injection of crude biological samples, with little pre-treatment (e.g. dilution with
internal standard), onto an LC-MS/MS system that incorporates switching valves
and multi-column configurations [85]. In its simplest form, bioanalysis using
column switching methods involves two columns, the first being used for sample
clean up (SPE) and the second for chromatographic separation [45]. Various
column dimensions can be configured for the fast analysis of drugs and their
metabolites in biological matrices at the ng/mL level or lower. There are minimal
sample preparation steps required; except for sample aliquoting, internal
standard addition and centrifugation [106,107]. These on-line SPE systems can
also be used in 96-well plate format in conjunction with a robotic liquid handling
system to provide complete automation. The pre-treated samples are then
injected directly onto the LC-MS/MS system auto-samplers with temperature
control capability to prevent evaporation and sample instability issues [107]. This
can provide a more environmental friendly analytical method which reduces the
Chapter 1
26
operator’s exposure to hazardous solvents, aerosols and toxins from the samples
and sample preparation process [108].
Several generic approaches have recently been developed for on-line
sample extraction coupled to LC-MS/MS [109]. These extraction phases include
restricted access media (RAM) [110,111], large-size particle disposable cartridges
and monolithic phases [88]. Zang et al. [82] described a novel on-line SPE
approach integrated with a monolithic HPLC column and triple quadrupole
tandem mass spectrometry for direct plasma analysis to simultaneously monitor
an eight-analyte test mixture in plasma. A simple column-switching
configuration that required only one six-port switching valve and one HPLC
pumping system allowed both isocratic and gradient separations for on-line SPE
LC-MS/MS. By using the monolithic column, the total analysis time was
significantly shortened while high chromatographic separation efficiency was
still maintained. This method provided the benefits of minimum sample
preparation, simplified hardware configuration, increased sample throughput,
efficient chromatographic capability, and a robust bioanalytical LC-MS/MS
system.
An alternative on-line sample preparation based on turbulent flow
chromatography (TFC) has been well-documented in recent years. A unique
characteristic of this on-line sample preparation approach is the use of narrow-bore
LC column (typically 1 mm x 50 mm) packed with large irregular shaped particles
(typically 30-50 m) as the stationary phase and relatively high flow-rates. The
large particles permit the use of extremely high linear velocities in the ranges of 3-5
mL/min. The combination of the high flow-rate and large particle sizes promotes
the rapid passage of the large biomolecules of the biological sample matrix with
the simultaneous retention of the small-molecule analytes of interest. This setup
Chapter 1
27
facilitates the sample extraction procedure without significant increases in column
pressure or blockages. Monolithic phases have been successfully employed in TFC
using high-flow on-line extraction. For example, Xu et al. described the use of a
short monolithic silica column for the bioanalysis of plasma and urine [112,113].
The performance of this extraction method was compared with that of an
automated liquid-liquid procedure; with both of the methods being found to give
similar performance.
Commercially available automated SPE systems, such as Symbiosis™, offer
the advantages of both off-line and on-line SPE. These SPE systems have
disposable cartridges contained in a 96-cartridge cassette and the use of multiple
cartridge trays permits more than 1000 samples to be run [81]. The Symbiosis™
system was described by Alnouti et al. [81] using both conventional C18 and
monolithic columns (ChromolithTM) for high-throughput direct analysis of
pharmaceutical compounds in plasma. The combination of on-line SPE with
monolithic columns enabled the development of high-throughput methods with 2
min total analysis time without compromising the data quality. Alternatively, these
systems can also be operated in an off-line mode either for method development or
routine SPE where the final elution sample is not compatible with the LC set-up
(i.e. the requirement for evaporation, buffer reconstitution followed by
chromatographic analysis). A multi-dimensional SPE mode allows the use of two
different extraction mechanisms on two separate cartridges in one sample run,
resulting in a more selective sample clean-up.
1.5 Conclusions and future possibilities
Monolithic materials provide both well-documented advantages and limitations.
Future bioanalytical interest in these materials could lie in further adaptations of
Chapter 1
28
the surface chemistry to select the right phases for isolation of specific analytes for
SPE, or to improve separations in HPLC. Once the phases are optimized for a
particular assay, other formats and scalability could be tested, with a view to
achieving miniaturize and high-throughput analyses. Pipette tip based formats
offer some exciting possibilities, especially when multi-purpose devices are used.
For example, enzyme digestion and selective SPE can be combined into a single
pipette tip using sequential layers of specific-purpose monolithic materials.
Furthermore, mixed mode adsorbents that combine ion-exchange and reversed-
phase characteristics for instance, can also provide some attractive options.
The use of electrospray nano-flow interfaces with LC-MS/MS platforms has
become far more prevalent, particularly in the peptide and biomarker
quantification arena. Difficulties in constructing suitable small-scale analytical
columns and preparative on-line clean-up columns have been encountered. The
latter can comprise specific immunoaffinity extraction columns, which can be
difficult to setup and integrate into a HPLC system. Most importantly, it can take a
relatively long time to identify and isolate suitable antibody reagents to prepare
the column. Small-scale monolithic columns could provide an alternative
approach, due to their flexibility and ease of preparation.
Capillary- and nano- LC formats are attractive both from the mass
sensitivity and environmental perspective. The trend in bioanalysis is leaning
strongly in this direction as samples are decreasing in size with the increased usage
of micro-sampling techniques, such as dried blood spot analysis. In this respect,
monoliths are likely to find increasing usage in areas such as isolating small
volumes for micro-sampling, tailored stationary phases and microchip-
based/micro-fabricated formats.
Chapter 1
29
1.6 Project aims
As presented in the literature review, monolithic materials, especially organic
polymer monoliths have been widely used for the separation and sample pre-
treatment process in bioanalysis. Organic polymer monoliths also possess
significant potential to be developed in different formats and functionalities, which
suggests their versatile applicability to all types of bioanalytical problems.
Taking into consideration the possibilities for future developments in this
field as identified in the literature review above, this project aims to explore ways
in which monoliths can be designed for use in high throughput absorption,
distribution, metabolism and elimination (ADME) assays. The focus in all of this
work will be on methods to miniaturize this analysis. The specific aims of the
project are to:
Investigate and synthesize of a range of functional polymer monoliths both
in bulk and in situ within capillaries and characterize the stationary phase
properties, such as porosity, morphology and ion-exchange functionality.
Test the suitability of the monolithic phases and optimize the selective
separation process of a set of target analytes of importance to ADME assays
using a capillary LC system.
Synthesize the optimized macroporous polymer monoliths in pipette tip
formats and explore the possibility of using trypsin immobilized enzyme
polypropylene pipette (IMEPP) tips for high throughput analyses.
Characterize and implement the developed IMEPP tips in a real
bioanalytical setting, i.e. for qualitative and quantitative analysis of target
proteins in biological matrix.
Synthesize these enzyme reactors in a range of different formats for
different bioanalytical settings.
Chapter 1
30
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Chapter 2
37
C h a p t e r 2
Experimental
This section describes the instrumentation, chemicals and procedures used
throughout this research, unless otherwise specified in a particular chapter.
2.1 Instrumentation
For the preparation of the monolith phases in situ within capillary and pipette tips,
an OAI deep UV illumination system (Model LS30/5, San Jose, CA, USA) fitted
with a 500W HgXe-lamp was used for UV exposure. For calibration, the irradiation
power was adjusted to 20.0 mW/cm2, using an OAI model 206 intensity meter with
a 260 nm probe head. The Teflon® -coated UV transparent fused-silica capillary (75
m I.D. x 375 µm O.D.) was obtained from Polymicro Technologies Inc. (Phoenix,
AZ, USA). Finntip-200 pipette tips consisting of homopolymeric isotactic
polypropylene (PP) were purchased from Pathtech Pty Ltd (Victoria, Australia).
Scanning electron microscopy (SEM) was performed on a DSM 962
scanning electron microscope (Zeiss, LEO) equipped with a DX-4 X-ray detection
system by EDAX. The SEM provides secondary and backscattered electron
imaging of biological, geological and other specimens yielding surface information.
SEM operated in low and high vacuum mode using accelerating voltages of 15, 20
and 25 kV.
The HPLC system used throughout this study was a Dionex Corporation
UltiMate® 3000 capillary LC system featuring an integrated flow-splitter connected
to a continuously monitored flow meter and control valve, which maintained
constant flow in the range of 0.1 to 10.0 L/min. It comprised a binary pump, a
Chapter 2
38
vacuum degasser, a column thermostat, an autosampler and a photodiode-array
UV detector. Chromeleon® Chromatography Management System version 6.80
was used for the system control, data acquisition and processing. OriginLab 8.0
software (Northampton, MA, USA) was also used for data analysis and graphing.
Direct infusion ESI-MS experiments were performed using a quadrupole
time-of-flight MS, Bruker MicrOTOF-Q MS controlled by a MicrOTOF-Q Control
software from Bruker Daltonics (Bremen, Germany). The liquid sample was
delivered via a KD Scientific (Holliston, MA, USA) syringe pump. The pump was
set at 180 µL/hr and an appropriate syringe from Hamilton (Reno, Nevada, USA)
was employed. Acquisition time was at least 2 min for each sample using the tune
wide LC method of the software.
For LC-MS separations, an Agilent 1200 Series LC system (Santa Clara, CA,
USA) consisting of a binary pump, a vacuum degasser, an autosampler, a
thermostatted column (30 °C) (Agilent Technologies, Palo Alto, CA) was coupled
to a Bruker micrOTOF-Q operating at a resolution of 10000 in the positive ion
mode. Argon was used as a nebulizing gas (1.5 bar) and nitrogen as a drying gas
(8.0 L/min, 200 °C). The capillary voltage was set to +4.0 kV. The reversed-phase
(RP) analytical column was a Dionex Acclaim® PolarAdvantge C16 (2.1 x 100 mm,
3 µm) coupled with binary RP solvents comprising (A) water and (B) acetonitrile,
both containing 0.1% formic acid. The data were collected and handled with the
Bruker Compass DataAnalysis 4.0 software.
Multiple reaction monitoring (MRM) transition development and
optimization were performed on an AB SCIEX Triple Quadrupole™ 5500 mass
spectrometer (Foster City, CA, USA) coupled to an Agilent 1100 binary HPLC
pump. An external Valco valve (10 or 6 port valves can be used) in “back flush
mode” was employed to control the column switching for both the loading and
Chapter 2
39
eluting gradient. A CTC PAL autosampler (Zwingen, Switzerland) fitted with a
250 µL syringe and 250 µL sample loop (for larger volume injection) was employed
for sample introduction. The system was controlled using Analyst version 1.5.1
which was also used for data acquisition and processing. An Onyx monolithic RP
C18 guard column (Torrance, CA, USA) and a Waters Xbidge BEH130 C18 column
(2.1 x 100 mm, 3.5 µm) (Milford, MA, USA) were used as pre-column and
analytical column in addition to the RP solvents were (A) 5% acetonitrile/ 95%
water and (B) 95% acetonitrile/ 5% water, both containing 0.1% formic acid.
Chromatography was carried out using a fast gradient; following a 2.5 min sample
loading, peptides were eluted over a 22 min gradient from 0-45% “B”. The column
was washed with 95% “B” for 1 min and re-equilibrated at initial conditions for 3
min prior to the next injection.
2.2 Reagents
Unless specified otherwise, the chemicals used were of analytical grade and are
listed in Table 2.1 to 2.6.
Chapter 2
40
Table 2.1: Chemicals used as buffers
Buffer Formula Supplier
Acetic acid CH3COOH BDH Chemicals
Formic acid HCOOH Sigma-Aldrich
Trifluoacetic acid CF3COOH Sigma-Aldrich
Disodium hydrogen phosphate Na2HPO4 Merck
Ammonium acetate CH3CO2NH4 Sigma-Aldrich
Ammonium bicarbonate CH3COONa BDH Chemicals
Tris(hydroxymethyl)aminomethane
hydrochloride
NH2C(CH2OH)3
· HCl
Sigma-Aldrich
Sodium dihydrogen phosphate NaH2PO4 Sigma-Aldrich
Hydrochloric acid HCl Sigma-Aldrich
Sodium hydroxide (99.99% and
99.0%)
NaOH Sigma-Aldrich
Chapter 2
41
Table 2.2: Chemicals used as analytes
Analyte Formula pKa Supplier
Arterenol,
hydrochloride
(HO)2C6H3CH(OH)C
H2NH2 · HCl 8.55 Sigma-Aldrich
Propranolol
hydrochloride C16H21NO2 · HCl 9.5 Sigma-Aldrich
Ketoprofen, C16H14O3 5.94 Sigma-Aldrich
Diclofenac C14H10Cl2NO2 4 Sigma-Aldrich
Ibuprofen C13H18O2 4.4 Sigma-Aldrich
Pindolol C14H20N2O2 9.04 Sigma-Aldrich
Acebutolol C18H28N2O4 9.2 Sigma-Aldrich
Chapter 2
42
Table 2.3: Chemicals used for methacrylate polymerization
Chemical Formula Supplier
-methacryloxypropyl-
trimethoxysilane (-MAPS)
C10H20O5Si Sigma-Aldrich
1,4-butanediol HO(CH2)4OH Sigma-Aldrich
1-propanol CH3(CH2)2OH Sigma-Aldrich
2,2-dimethoxy-2-
phenylacetophenone
(DMAP)
C6H5COC(OCH3)2C6H5 Sigma-Aldrich
Methanol CH3OH Sigma-Aldrich
Cyclohexanol C6H12O Merck
Decanol C10H22O Sigma-Aldrich
Dodecanol C12H26O Sigma-Aldrich
Basic alumina activity
grade I, Type WB-2
Al2O3 Sigma-Aldrich
Butyl methacrylate (BMA) H2C=C(CH3)CO2-(CH2)3CH3 Sigma-Aldrich
3-Sulfopropyl methacrylate
(SPMA)
H2C=C(CH3)CO2(CH2)3SO3K Sigma-Aldrich
Ethylene glycol
dimethacrylate (EDMA)
[H2C=C(CH3)CO2CH2]2 Sigma-Aldrich
Methyl methacrylate CH2=C(CH3)COOCH3 Sigma-Aldrich
2-Hydroxyethyl
methacrylate (HEMA) H2C=C(CH3)COOCH2CH2OH Sigma-Aldrich
Azobisisobutyronitrile
(AIBN)
(CH3)2C(CN)N=NC(CH3)2CN Sigma-Aldrich
Chapter 2
43
Table 2.4: Protein samples used for studies
Protein Molecular Weight (Da) Source Supplier
Trypsin 23800 Bovine
pancreas
Sigma-Aldrich
Melittin 2846.5 Bee venom Sigma-Aldrich
Calcitonin gene
related peptide
3806.3 Rat Sigma-Aldrich
Cytochrome c 11701.5 Equine heart Sigma-Aldrich
Myoglobin 16951.4 Equine heart Sigma-Aldrich
Transferrin 77050 Human Sigma-Aldrich
Serum albumin 66000 Bovine Sigma-Aldrich
Polyclonal IgG 150000 Human Sigma-Aldrich
Table 2.5: Organic solvents used in this work
Chemical Formula Supplier
Acetone (CH3)2CO BDH Chemicals
Acetonitrile CH3CN Merck
Chapter 2
44
Table 2.6: Other chemicals used in this work
Chemicals Formula Supplier
Urea NH2CONH2 Sigma-Aldrich
Guanidine hydrochloride (GdnHCl) CH5N3 · HCl Sigma-Aldrich
Iodoacetamide (IAA) ICH2CONH2 Sigma-Aldrich
DL-Dithiothreitol (DTT) HSCH2CH(OH)CH(OH)C
H2SH
Sigma-Aldrich
Methoxy polyethylene glycol 550
methacrylate (PEGMA)
H2C=C(CH3)CO2(CH2CH
2O)nCH3
Sartomer
Tris(2-carboxyethyk)phosphine
hydrochloride (TCEP)
C9H15O6P · HCl Sigma-Aldrich
4,4-Dimethyl-2-vinyl-2-oxazolin-5-
one (VAL)
C7H9NO2 Tokyo Chemical
Industry Co.
Benzophenone (C6H5)2CO Sigma-Aldrich
Copper(II) sulfate pentahydrate CuSO4.5H2O Sigma-Aldrich
Potassium chloride KCl Sigma-Aldrich
Ethanolamine NH2CH2CH2OH Sigma-Aldrich
Chapter 2
45
2.3 Procedures
2.3.1 Buffer and standard preparation
Buffers, analyte and protein standards were prepared with water purified by a
Milli-Q water purification apparatus (Millipore, Bedford, MA, U.S.A.). All buffers
were degassed using vacuum sonication and filtered with a Millex-HA 0.45 μm
disc filter (Millipore). Monomers were purified prior to use by passage through a
bed of basic alumina (Brockman activity I, 60-325 mesh), followed by distillation
under reduced pressure. Distilled monomers were stored at -20 °C in the freezer.
2.3.2 Sample injection
For the HPLC separations, samples were injected on to the monolithic column via
the HPLC autosampler. The capillary HPLC column temperature was maintained
at 40 °C. The mobile phases A and B consisted of 0.1% TFA in water (v/v) and 0.1%
TFA in acetonitrile (v/v), respectively. For all samples, the injected volume was 1
L. Preliminary UV analyses were performed at several different wavelengths for
each drug sample, so as to select the optimum wavelength for all the drug analytes
and utilize a single wavelength UV detector optimally.
For the LC-MS separations, samples were injected on to the column via the
Agilent 1200 Series autosampler.
For the Agilent 1100/ AB SCIEX Triple Quadrupole™ 5500 mass
spectrometer, samples were introduced by CTC PAL auto sampler fitted with a 250
µL syringe and 250 µL sample loop.
2.3.3 Surface modification in fused-silica capillaries
In order to anchor the monolithic structure to the capillary wall by covalent
bonding, the fused-silica capillaries were surface-modified using the procedure of
Schaller et al. [1]. The Teflon® -coated UV transparent fused-silica capillaries were
Chapter 2
46
rinsed using a Harvard syringe pump (Harvard Apparatus, Holliston, MA, USA)
and a 250 L gas-tight syringe (Hamilton Company, Reno, NE, USA) with acetone
and water, activated with 0.2 mol/L NaOH for 30 min, washed with water, then
with 0.2 mol/L HCl for 20 min, rinsed with water and ethanol. A 20% (w/w)
solution of -methacryloxypropyl-trimethoxysilane (-MAPS) in 95% ethanol
adjusted to pH 5 using acetic acid was pumped through the capillaries at a flow-
rate of 15 L/hr for 1 h. The capillary was then washed with acetone and dried with
a stream of air and left at room temperature for 24 h.
2.3.4 Synthesis of polymer monoliths in capillaries
The monomers (butyl methacrylate and sulfopropyl methacrylate), cross-linker
(ethylene glycol dimethacrylate), porogens (1,4-butanediol and 1-propanol) were
mixed with the UV initiator 2,2-dimethoxy-2-phenylacetophenone (DMAP) to give
a clear, organic solvent mixture. This mixture was sonicated for 10 min and
subsequently degassed with nitrogen for 5-10 min prior to UV-initiated
polymerization. The overall ratio of monomer to porogen was held constant. Only
the relative composition of the porogenic solvents was varied to give a controlled
porous structure. A small portion of polymerization mixture was pumped into a
fused-silica capillary which had previously been derivatized with -MAPS. The
ends of the capillary were plugged with rubber septa. The capillary containing the
polymerization mixture was irradiated under constant UV irradiation for 10 min
using the OAI deep UV illumination system described in Section 2.1. The resultant
monolithic column was flushed with methanol for 5 h at 1200 kPa, followed by
water using a syringe pump, prior to the HPLC separations.
2.3.5 Measurement of ion-exchange capacity
The ion-exchange capacity of the monolithic stationary phases synthesized in situ
in the capillary was determined by copper adsorption/desorption using the liquid
Chapter 2
47
chromatography system (Dionex). The capillary columns were first loaded with
potassium using 0.2 mM potassium chloride solution. Excess chloride was flushed
from the column using MilliQ water at a rate of 0.2 μL/min for a period of 30 min.
The capillary was disconnected from the pump and the system was flushed with
0.1 mM copper(II) sulfate pentahydrate (CuSO4.5H2O) solution for a period of 30
min using a flow-rate of 2 µL/min. The capillary column was reconnected to the
system and 0.1 mM copper solution was pumped through the capillary at a rate of
0.2 µL/min. The absorbance of the eluent was monitored at 235 nm until
breakthrough was observed and the absorbance reached a stable value. The
process was further repeated twice.
2.3.6 Porosimetry
Porosimetry measurements were performed by mercury intrusion (Micrometrics
Pore Sizer 9310, Norcross, Georgia, USA), using a 3 cc glass penetrometer designed
for powder analysis. Characterization measurements were conducted on 0.1 g of
polymer material placed into the penetrometer head, before securely fastening the
lid with a nut and screw cap. Pressure measurements were made from levels
between 0.5 psi and 30,000 psi (2,000 bars), yielding pore size distributions in the
range of 0.003 to 360 µm.
2.3.7 Surface modification in polypropylene pipette tips
Finntip pipette tips (Finntip-200) consisting of homopolymeric isotactic
polypropylene (PP) [3] were used in this study. The surface modification in pipette
tip was done by using both single- and two-step photografting according to the
procedure described previously [3] (Figure 2-1). The photoinitiator benzophenone
was used to generate radicals at the surface of PP by hydrogen abstraction (Figure
2-1). For single-step photografting, a stock solution containing methyl methacrylate
(MMA) (0.485 g) and ethylene glycol dimethacrylate (EDMA) (0.485 g) with a ratio
Chapter 2
48
Figure 2-1: Schematic diagram of the single- and two-step photografting procedures.
Chapter 2
49
(1:1) (% w/w) was prepared. From this stock solution, a modification mixture
containing MMA:EDMA:BP (benzophenone) (3% w/w) (0.03 g) was prepared.
Before use, the stock solution was vortexed and purged with nitrogen for 10 min to
remove oxygen. The Finntip pipette tips were then filled with 60 µL modification
mixture and sealed with parafilm [Figure 2-2 (a)]. The filled pipette tips (with
tapered end sealed with parafilm) were irradiated on both sides of the tips under
UV light for 12 min (or 10.5 min if EDA was used). Once the reaction was
complete, the pipette tip was washed with methanol and acetone, before drying in
a vacuum oven at room temperature for 10 min. For the two-step surface
modification procedure, BP was first grafted to the surface of PP tips by filling the
tips with 3% (w/w) BP solution in methanol and sealed with parafilm. The filled tip
was irradiated under UV for 12 min. After photgrafting BP, the tip was rinsed with
methanol and dried. Next, the tip was filled with the monomer mixture (MMA and
EDMA) and irradiated under UV. Upon completion of the reaction, the pipette tip
was washed with methanol and acetone, and was then dried in a vacuum oven at
room temperature for 10 min.
Polypropylene tip Surface modification mixture:(1:1 methyl methacrylate: ethylene glycol dimethacrylate + 3% benzophenone)
Free double bonds
UV
(a) (b)
Figure 2-2: Schematic diagram of the photoinduced surface modification and
preparation of a monolith in an empty pipette tip. (a) Tip is filled with a BP
solution in methacrylate and irradiated with UV source; (b) a grafted
compatibilizing polymer layer containing free double bond is created at the
surface.
Chapter 2
50
2.3.8 Preparation of porous polymer monoliths in PP tips
A polymerization mixture consisting of 16% (w/w) BMA, 24% (w/w) EDMA, 12%
(w/w) 1,4-butanediol, 42% (w/w) 1-propanol and 1% (w/w) DMAP (with respect to
monomers) was prepared and purged with nitrogen for 10 min. The surface-
modified pipette tip was filled with 20 µL of the homogenous polymerization
mixture, and irradiated using an OAI deep UV illumination system. The
polymerization was allowed to proceed first for 40 min with the sharp end of the
tip down, and then for 25 min with the sharp end up (Figure 2-3), resulting in a 1
cm monolith support in situ polypropylene pipette tip. After photopolymerization,
the pipette tip with polymer monolith was then washed with methanol for 1 h
using the vacuum filtration apparatus (Figure 2-4), to allow the solvents to flow
through the porous monolith. The pore size of the monolith was ~ 2.8 µm, as
measured by mercury intrusion porosimetry [4].
2.3.9 Digestion protocol for immobilized enzyme polypropylene
pipette (IMEPP) tip and MonoTip® Trypsin
The pipetting procedure of the IMEPP tip was done by attaching silicone tube to a
curved tip syringe (Monojet 412, Figure 2-5). The IMEPP tip was equilibrated by
aspirating and dispensing the digestion buffer for 10 cycles. The protein sample
solution was heated in a dry bath incubator at 37 C prior to the digestion by
IMEPP tip. Then, protein sample was aspirated and dispensed (20 cycles). The
pipette tip was removed from the syringe and soaked with the sample solution for
30 min. The tip was removed and reconnected to the syringe to dispense the
remaining digestion solution at the tip end. After the digestion procedure was
complete, the solution with peptides was protonated by adding formic acid in
order to achieve a final concentration of 0.1%. The same digestion procedure was
used for the MonoTip® Trypsin except that an autopipette was used instead of the
Chapter 2
51
Figure 2-3: Schematic diagram showing in situ preparation of polymer monoliths in polypropylene pipette tip.
Chapter 2
52
Figure 2-4: A vacuum filtration apparatus was assembled using a filter flask with
a side arm which permits making a connection to vacuum pump. The top
opening of the suction flask accommodates a two-hole rubber stopper which in
turn supports a glass tube specifically used for suction operation for pipette tips.
Chapter 2
53
Figure 2-5: Silicone tube attached to curve syringe (Monojet 412) for pipetting
IMEPP tip.
Chapter 2
54
curved tip syringe.
2.3.10 Scanning electron microscopy
The monolithic capillary was cut into 1 cm fragments that were mounted
perpendicularly to a 12 mm pin-type aluminium stub using epoxy resin. High-
resolution images were obtained by coating the capillary with gold or platinum
(nominally 40 nm thick). In order to take cross-sectional SEM images of the pipette
tips, samples approximately 3 mm in length were cut from the tip with a razor
blade and mounted on an aluminum stub. This was coated with a thin layer of
platinum using an EIKO IB5 high-resolution platinum coater.
2.4 Calculations
2.4.1 Permeability based on Darcy’s Law
The flow of solution through a porous medium is directly proportional to the
pressure drop over a given distance. Using this relationship, the permeability B0,
which represents the resistance to mobile phase flow through the monolithic
column can be calculated by pumping five different solvents through the column
at different linear flow-rates according to [4] where B0 is the column permeability, υ
is the linear velocity (m/min), L is the column length (m), η is the dynamic solvent
viscosity (Pa·s) and ΔP is the column back pressure (Pa).
LB0
2.4.2 Retention factors
Retention factors were calculated using the following expression:
0
0
t
ttk R
Chapter 2
55
where Rt is the elution time of the analyte and 0t the elution time of the flow
marker.
2.4.3 Sequence coverage
Sequence coverages were calculated using the following expression:
%1002
1..
P
PCS
where P1 is the number of peptides identified, P2 is the expected number of
peptides after protein digestion. Sequence coverages of over 80% are very useful
and important for proteomic studies.
2.5 References
[1] D. Schaller, E.F. Hilder, P.R. Haddad, Anal. Chim. Acta 556 (2006) 104.
[2] D.S. Peterson, T. Rohr, F. Svec, J.M. Frechet, Anal. Chem. 75 (2003) 5328.
[3] Z. Altun, A. Hjelmstroem, M. Abdel-Rehim, L.G. Blomberg, J. Sep. Sci. 30
(2007) 1964.
[4] Z. Jiang, N.W. Smith, P.D. Ferguson, M.R. Taylor, Anal. Chem. 79 (2007)
1243.
Chapter 3
56
C h a p t e r 3
Mixed-Mode Porous Polymer Monoliths for
Separation of Small Molecule Pharmaceuticals
3.1 Introduction
The increasing cost of drug discovery and development places a significant burden
on the drug candidate evaluation process. It has been estimated that discovering
and bringing one new drug to the market takes an average of 14 years of research
and clinical development efforts, and costs around 2 billion US dollars. Of ten
thousand or more drugs tested in early drug discovery, only one may eventually
lead to a drug available to the market. Therefore, high-throughput adsorption,
distribution, metabolism and excretion (ADME) assays for compound drug
development properties are extremely important in this process [1].
In recent years, monolithic supports as stationary phases in HPLC have
gained significant interest in pharmaceutical analysis, in particular for small
molecules as well as protein and peptide separations in gradient and isocratic
modes [2-4]. The most prevalent hyphenated techniques, liquid chromatography
coupled to tandem mass spectrometry, have become critical tools in
pharmacokinetics and metabolism studies at various drug development stages [5].
Tandem mass spectrometry based methods provide unique analytical sensitivity,
selectivity and speed for monitoring one specific target compound in biological
samples. This has reduced the need for high resolution power in LC analysis.
Despite only acceptable resolution being required in the sample analysis, certain
sample preparation steps are needed to remove endogenous interference species
Chapter 3
57
from the matrix prior to the HPLC-MS/MS assay for small molecules. This
prevents the HPLC column from clogging in reversed-phase (RP) chromatography
and to avoid ion source contamination in the mass spectrometer [5]. In addition,
the use of capillary columns with a nanospray interface gives better compatibility
and sensitivity with mass spectrometry. Other advantages of the capillary column
format include the small consumption of both sample and solvents and providing
fast assays and short method development timelines [6]. Therefore, an increasing
number of researchers are focusing on improving the capacity, selectivity and
repeatability of columns in miniaturized formats [7-9].
Monolithic materials can be divided into two main categories, namely silica-
based (inorganic) and rigid polymer (organic) based monoliths. The silica-based
monoliths can be prepared by a sol-gel method [10] and the rigid polymer-based
monoliths are generally made by in situ polymerization of monomers, cross-linkers
and porogens [11]. Polymer-based monoliths have been widely used as matrices
for sample preparation and separation since being first introduced by Hjerten et al.
[12] and following further innovations by Svec and Fréchet [13]. Although there
are some concerns of using polymer-based monoliths for sample preparation, such
as low surface area and the relatively poor chromatographic performance for small
molecules [6,14,15], the advantages of these materials clearly outweigh the silica-
based monoliths in terms of the pH stability and a wealth of surface chemistries
available [14]. Monolithic materials also offer particular advantages for high-
throughput ADME assays due to high permeability (i.e. high flow-rates can be
used) and excellent column stability [9,16].
Mixed-mode strong cation-exchange (SCX) and reversed-phase (RP)
materials are the most often used modes of chromatography for the extraction and
separation of different types of compounds of pharmaceutical interest [17]. These
Chapter 3
58
approaches have been used for over 80% of the separation of target analytes in
pharmaceutical analysis [18]. Several approaches have been reported to synthesize
strong SCX polymer monoliths containing sulfonic acid groups, including
adsorption [19], post-modification [20], and copolymerization [21]. Recently,
monolithic columns with mixed-mode of strong anion-exchange (SAX) and
hydrophilic (HI) or RP interactions have also been reported by several groups
[20,22,23]. The mixed-mode monolithic columns with SAX interaction were used to
counter undesirable electrostatic adsorption between positively charged basic
compounds and the stationary phases [24,25]. These columns can be prepared
using a monolithic matrix containing active groups followed by their
functionalization with versatile surface chemistries [20,26]. However, the post-
functionalization strategy with multiple steps can be time-consuming and precise
control over the number of ionizable functionalities can be difficult to achieve. An
alternative approach to obtain ion-exchange functionality monolithic columns is
the direct incorporation of ion-exchange functional monomers into continuous
polymer matrices by the co-polymerization of suitable monomers and cross-linkers.
For example, Lin et al. [22] reported a novel a mixed-mode SAX-HI stationary
phase for the separation of polar-charged nucleotides and neutral compounds.
Jiang et al. [17] fabricated a SCX-RP monolithic columns for the separation of basic
compounds using micro-HPLC. Both works have demonstrated the fast
preparation procedures of mixed-mode functionality monolithic columns and it is
easy to control the average concentration of functional groups in the monolith,
thus, more repeatable and manufacturable.
Despite the enormous significance for users, only a few studies relating to
the repeatability and stability of polymer-based monolithic columns are found in
the literature [9,27]. Geiser et al. [9] used three standard proteins – ribonuclease A,
Chapter 3
59
cytochrome c and myglobin to investigate the stability and repeatability of
poly(BMA-co-EDMA) capillary columns. They found excellent repeatability of
retention times for the reparation of three proteins as evidenced by percent relative
standard deviation (%RSD) values of less than 1.5%. The stability of retention times
was also monitored and no significant shifts in either retention times or
backpressure were observed after more than 2200 protein separations. Recently, Li
et al. [28] reported a highly cross-linked network resulting from single crosslinking
monomers that improved column-to-column repeatability, better mechanical
stability and higher surface area of polymeric monoliths [14]. However, this
polymer monolith prepared by a single monomer does not make it easily possible
to provide mixed-mode functionality. To date, there are also only a few studies on
the stability and repeatability of mixed-mode SCX-RP functionalities polymer-
based monolithic columns for capillary LC [17,22]. Further effort is therefore
needed to develop novel stationary phases with negatively charged and RP
functionalities for highly efficient separation of polar analytes. In this regard, we
report on the characterization, repeatability and stability of a mixed-mode SCX-RP
polymeric monolithic stationary phase prepared by the copolymerization of a
functional monomer, namely 3-Sulfopropyl methacrylate (SPMA) with BMA and
EDMA within the confines of 75 µm i.d. fused-silica capillaries. The impact of
different polymerization parameters (monomer content, porogenic solvent and
cross-linker) on the porous properties, and hence on the separation selectivity for
some acidic drugs and -blockers, was studied.
3.2 Experimental
The general experimental details are described in Chapter 2. Detailed conditions
are elaborated in each of the figure captions.
Chapter 3
60
3.2.1 Preparation of porous polymer monoliths in fused-silica
capillaries
The surface modification in fused-silica capillaries was done using the procedure of
Schaller et al. [29]. The short (~10-15 cm in length) surface-modified capillary was
filled by capillary action with the degassed polymerization mixture consisting of
monomers, porogens together with 1% (w/w) DMAP (with respect to monomers).
Several SPMA monoliths that consist of similar composition and percentages of
monomers but differ in the composition of porogens were prepared (S1-S7), as
described in Table 3.1. In addition, monoliths (S1M-S3M) consisting of a greater
amount of cross-linker (EDMA) than the functional monomer (BMA) were
prepared, as shown in Table 3.2. The short columns filled with the polymerization
mixture were placed under the light source and irradiated with UV light for 10 min
at a distance of 30 cm. The unreacted porogens were removed from the short
monolithic columns by pumping the columns with methanol using a syringe pump
at a flow-rate of 100 µL/hr for 4 h before being conditioned with water and mobile
phase, both for 1 h at 30 µL/hr.
3.2.2 Bulk monolith preparation
Bulk polymers were prepared using a sandwich container (Figure 3-1) for
characterization studies. The sandwich container is made of stainless steel and has
a diameter of 6.35 cm. It consists of two halves; a base with a thickness of 1 cm and
an upper rim which is 0.45 cm thick. The central part of the base is made of
polypropylene and there is a shallow cavity which has a diameter of 3.5 cm and a
depth of 2 mm. For the monolith formation, the polymerization mixture was
injected into this shallow cavity, and a piece of quartz plate of 4.6 cm in diameter
and 2 mm in thickness was placed in between the two halves of the container. The
two halves were fastened together with three screws that are at 120 degrees from
Chapter 3
61
Table 3.1: Effect of variation of the percentage of porogenic solvents on the pore
size of the studied monoliths (S1-S7) prepared from a polymerization mixture
consisting of 0.4% (w/w) SPMA, 23.6% (w/w) BMA and 16% (w/w) EDMA that
constitute 40% (w/w) of the polymerization mixture with respect to 60% (w/w)
porogenic solvents.
Polymer
1,4-butanediol
[% (w/w)]
1-propanol
[% (w/w)]
Water
[% (w/w)]
Pore size
[µm]
S1 30 60 10 2.70
S2 20 70 10 2.20
S3 10 80 10 0.14
S0 0 90 10 NA
S4 90 0 10 NA
S5 80 10 10 NA
S6 70 20 10 1.06
S7 60 30 10 1.36
NA – Polymer does not form for measurement.
Chapter 3
62
Table 3.2: Effect of variation of the percentage of porogenic solvents and alcohol
chain length on the pore size of the studied monoliths (SMeth-SL) prepared
from a polymerization mixture consisting of 0.4% (w/w) SPMA, 23.6% (w/w)
BMA and 16% (w/w) EDMA that constitute 40% (w/w) of the polymerization
mixture with respect to 60% (w/w) porogenic solvents (percentage shown in
table).
Polymer*
1,4-butanediol
[% (w/w)]
Alcohol
[% (w/w)]
Water
[% (w/w)]
Pore size
[µm]
S1Meth 30 60 10 1.23
S2Meth 20 70 10 1.68
S3Meth 10 80 10 2.24
S1C 30 60 10 1.71
S2C 20 70 10 0.97
S3C 10 80 10 0.07
S1D 30 60 10 1.90
S2D 20 70 10 NA
S3D 10 80 10 NA
S1L 30 60 10 2.07
S2L 20 70 10 NA
S3L 10 80 10 NA
NA – Polymer does not form for measurement.
*Meth: methanol (C1)
C: cyclohexanol (C6)
D: decanol (C10)
L: dodecanol (C12)
Chapter 3
63
one another. The polymerization mixture was inserted via a syringe fitted with a 25
gauge syringe needle in the sandwich container. With the solution in place and the
two halves of the sandwich container secured, the container was then irradiated
under UV for 20 min. For characterization of the porous properties, the polymer
material was removed from the sandwich container and the material was extracted
with methanol using Soxhlet apparatus for 12 h, before being vacuum-dried at 60
°C for a further 12 h. These materials were later used for the determination of pore
size distribution and pore volume.
3.2.3 Standard solutions and sample preparation
Drug samples arterenol, propranolol and ketoprofen, diclofenac and ibuprofen
were prepared in water and methanol at 1 mg/mL. All were further diluted or
multicomponent standard mixtures of samples were made using ultra-pure water
from the Milli-Q Element system (Millipore, Billerica, MA, USA).
Figure 3-1: Sandwich container used for bulk monolith preparation.
Chapter 3
64
3.3 Results and discussion
3.3.1 Preparation of polymer monoliths
Several prerequisites should be met when designing a macroporous monolithic
polymer for use in separation media and solid-phase extraction (SPE). Among
these, material surface chemistry, porosity, pore size distribution and rigidity
constitute the core of the monolith formation strategy [14]. In the monolithic
systems studied here, the first two variables depended on the type of monomers
used. SPMA was used as a functional monomer for its strong cation-exchange
properties, EDMA was used as a crosslinking monomer and BMA monomer was
used as a functional monomer with RP properties. Since the other variables are
closely related to the porous structure of the monolith, these depend on the pore-
forming solvent (porogens). Consequently, the number of variables that were
used for the control of porous properties in this system was limited to: a) the
ratio of monomers to pore-forming solvents in the polymerization mixture, b) the
percentage of different solvents in the pore-forming mixture and c) the ratio of
cross-linker to functional monomers in the polymerization mixture.
SPMA-based monolithic fused-silica capillary columns with 75 µm
internal diameter and varying pore properties were prepared in situ by a UV-
initiated free-radical copolymerization reaction. The mixed-phase monomers
consisted of sulfopropyl methacrylate SPMA [0.4% (w/w)], butyl methacrylate
BMA [23.6% (w/w)] and ethylene dimethacrylate EDMA [16% (w/w)], with 1,4-
butanediol, 1-propanol and water as porogenic solvents, and 2,2-dimethoxy-2-
phenylacetophenone (DMAP) as the photo initiator. As the porosity of
monolithic phases can be varied by making minor alterations to the composition
of the polymerization mixture, the total monomer concentration in the prepared
phases was kept constant at 40% (w/w) with respect to the total porogen solvents
Chapter 3
65
consisting of 1,4-butandiol, 1-propanol and water at 60% (w/w). Thus, only the
percentage of solvents in the total porogenic solvent composition, the percentage
of cross-linker to functional monomers, and their effects on the pore size and
separation of different compounds were studied. For comparison purposes, the
alkyl chain of the alcohol in the porogenic solvent was altered in a separate
experiment. Thus, either methanol (C1), cyclohexanol (C6), decanol (C10) or
dodecanol (C12) was used to replace the 1-propanol in the aforementioned
polymerization mixture.
Three types of polar porogen mixtures that favor early phase separation of
the polymer were used for the creation of the porous structures. Thus, 1-propanol,
1,4-butandiol and water that constituted 60% (w/w) of the polymerization mixture
were tested. Out of the 60% (w/w) of total porogenic solvents, the amount of water
[10% (w/w)] was fixed while the ratio of 1,4-butanediol to 1-propanol was
systematically varied in the total porogen solvents in an attempt to prepare the first
set of capillaries, namely S1 to S7 with different properties (Table 3.1).
Furthermore, the effect of alkyl chain length in the alcohol porogenic solvent
mixture on the pore size and separation of the investigated drugs was studied.
Thus, 1-propanol was replaced with methanol (S1meth-S3Meth), cyclohexanol
(S1C-S3C), decanol (S1D-S3D) or dodecanol (S1L-S3L) using similar percentages of
porogenic solvents as reported above (Table 3.2).
3.3.2 Characterization of SPMA-based polymer monoliths
3.3.2.1 Mercury intrusion porosimetry
Monoliths prepared in situ in capillaries do not have sufficient bulk for
porosimetry measurements. Therefore, polymerization was carried out in a
sandwich device of larger volume to obtain sufficient amount of monolithic
polymer so that the mercury intrusion porosimetry measurements on bulk
Chapter 3
66
material can be representative of the porous properties [30]. This technique
revealed the median pore diameters from all the prepared monoliths, with the
value of the median pore diameter being 2.70 µm and 2.20 µm for S1 and S2,
respectively. The porogenic solvent composition of S1 and S2 consisted of 1,4-
butandiol, 1-propanol and water, in the ratios of 30:60:10 and 20:70:10 (%
w/w/w), respectively. On the other hand, the pore diameters of S3, S6 and S7
were 0.14 µm, 1.06 µm and 1.36 µm, respectively, and their corresponding
porogenic solvent composition ratios were 10:80:10; 70:20:10; and 60:30:10 (%
w/w/w), respectively (Table 3.1).
For S1 and S2, early phase separation was achieved and larger pore size
was obtained. However, the relatively smaller pore sizes of S3, S6 and S7 were
probably due to the simultaneous decrease in solubility of both the monomers
and the polymers in the system which contained a certain percentage of
porogenic solvents. When the amount of 10% (w/w) 1,4-butanediol in
combination with 80% (w/w) 1-propanol was used as in S3, 70:20 [1,4-
butanediol:1-propanol, % (w/w)] as in S6, or 60:30 [1,4-butanediol:1-propanol, %
(w/w)] as in S7, the solubility of both monomers and the polymers decreased
(Table 3.1). The polymer chains remained soluble in the mixture for a longer
period prior to phase separation due to the absence of contrasting polarity within
the separated nuclei and also in the surrounding solution. Monomers were not
compelled to adsorb preferentially into the nuclei and the polymerization
proceeded with the formation of nuclei that remained individualized.
Consequently, a larger number of individual nuclei competed for the remaining
monomers [31], and this led to a large number of small microglobules that
aggregated to form macroporous structure with fine individual microglobules
and small pore sizes (e.g. S3, S6 and S7) (Table 3.1). On the contrary, a decrease in
Chapter 3
67
the solubility of the polymer formed in a system containing 30% (w/w) 1,4-
butandiol relative to that of 60% (w/w) 1-propanol, as in S1, led to an early phase
separation. The separated nuclei, swollen with 1,4-butandiol (a more polar
solvent than 1-propanol) preferentially extract the monomers from the
surrounding liquid polymerization containing the less polar 1-propanol since the
monomers favor the more polar environment. The enlarged nuclei could attract
and coalesce with the newly precipitated chains and further increase their size,
thus leading to the formation of large microglobules and larger pores. This
explanation is in agreement with the previously reported pore formation
mechanism [31]. The homogenous pore size distribution curve of S1, S2 and S3
measured by mercury intrusion porosimeter is shown in Figure 3-2. Additionally,
S0, S4 and S5 with 0:90, 90:0 and 80:10 [1,4-butandiol:1-propanol, % (w/w)] and a
fixed 10% (w/w) water formed an emulsion in the polymerization mixture due to
poor solubility of monomers (Table 3.1).
On the other hand, by varying the alkyl chain in the alcohol porogenic
solvent from 1-propanol (C3) to methanol (C1), cyclohexanol (C6), decanol (C10) or
dodecanol (C12), an increase in pore size was also observed from 1.23 µm for
methanol (S1Meth) to 2.07 µm for dodecanol (S1L) (Table 3.2). In addition, the
increase of methanol percentage relative to that of 1,4-butandiol in the porogenic
solvents led to an increase in the pore sizes of the formed monoliths (Figure 3-3
and Table 3.2). This is in contrast with S1-S3 where the pore sizes decreased when
increasing the amount of 1-propanol compared to methanol (Tables 3.1 and 3.2,
Figure 3-3). No monolithic polymer formed for S2decanol, S3decanol, S2dodecanol
and S3dodecanol due to solubility problems. Based on these results, monoliths
prepared with different alcohol alkyl chains were not further considered; only
monoliths prepared with 1-propanol (S1-S3, S6 and S7) were investigated further.
Chapter 3
68
Figure 3-2: Pore size distribution curve of S1, S2 and S3 measured by mercury
intrusion porosimeter.
Figure 3-3: Relationship between median pore diameter and amount of alcohol
porogen.
Chapter 3
69
3.3.2.2 Scanning electron microscopy (SEM)
Scanning electron microscopy (SEM) was initially used to study the effect of
variation of the weight percentage of the porogenic solvent on the morphology of
the prepared monoliths. This was achieved by comparing polymers prepared in
bulk and in capillary. Both showed similar SEM images regardless of the method
of preparation (Figure 3-4).
The morphology of the prepared monoliths S1, S2, S6 and S7 showed that
the copolymerized monolith was composed of a heterogeneous surface of spherical
units agglomerated into larger clusters interdispersed by large-pore channels, a
characteristic sign of monolithic structures (Figure 3-4). S3 with 10:80 1,4-butandiol
to 1-propanol [% (w/w)] and a fixed 10% (w/w) of water was not permeable when
a mobile phase of 50:50 acetonitrile:water [% (w/w)] was used. It was observed to
be a more gel-like monolith rather than having macropores such as exhibited by
the S1, S2, S6 and S7 monoliths (Figure 3-5).
(a) (b)
Figure 3-4: SEM images of S1 prepared (a) in situ in capillary and (b) in bulk
process.
Chapter 3
70
Figure 3-5: SEM images of S1, S2, S3, S6 and S7 prepared by in situ polymerization process showing an obvious feature
of the effect of 1-propanol on the morphology of SPMA-based monolith prepared in capillary column.
Chapter 3
71
3.3.3 Chromatographic characterization of SPMA monolithic
columns
3.3.3.1 Separation mechanism
The main interest in this part of the study was the development of new SCX-RP
functionality polymer-based monolithic columns that could tolerate relatively large
injection volumes and exhibit adequate separation performance of small molecules.
The desired material would also have the capability for upscaling for preparative
chromatography or downscaling for low resolution chromatography (e.g. rapid
screening) or sample preparation applications. Therefore, 1 µL of sample volume
was injected for all the chromatographic evaluations. By mixing SPMA (cation-
exchange functional monomer) and BMA (RP functional monomer) the formation
of a mixed-mode SCX-RP material resulted.
3.3.3.1.1 Reversed-phase interaction
Monolithic columns with RP retention mechanisms have been widely used for the
analysis of various complex samples [32]. The hydrophobicity of the stationary
phase determines the selectivity of the separation, and RP retention can be easily
controlled by adjusting either the percentage of the organic modifier in the mobile
phase or the hydrophobicity of the surface [33]. In order to confirm a RP separation
mechanism of the prepared mixed-mode monolith, three acidic drugs (i.e.
diclofenac, ibuprofen and ketoprofen) were chosen as test compounds in the
capillary LC mode. At low pH, these drugs existed as neutral analytes and were
separated based on RP interaction. The logarithm of the retention factor of the
target analytes was plotted against volume percentage of the organic solvent
(acetonitrile) in the mobile phase. As shown in Figure 3-6, as the volume of organic
modifier in the mobile phases was increased, the retention for the investigated
Chapter 3
72
Figure 3-6: Plot of logarithm of the retention factor of a set of acidic drugs
versus logarithm of the organic solvent in the mobile phase acetonitrile:water
with 0.1% trifluoroacetic acid (TFA) separated on S1 monolithic column.
Capillary 12 cm x 75 µm i.d. Conditions: flow-rate, 2 µL/min; 1 µL injection
volume; column temp, 40 °C; detection wavelength, 219 nm.
Chapter 3
73
analytes decreased. The linearity of these plots confirms the RP nature of the
separation mechanism.
3.3.3.1.2 Cation-exchange interaction and capacity
The inclusion of SPMA provides cation-exchange sites in the monolith. The ion-
exchange contribution to the separation mechanism was also investigated. Ion-
exchange interactions between the charged analytes and the stationary phase can
be influenced by the concentration of the competing ions in the mobile phase used.
A competing ion (NH4+) was chosen and its concentration was altered, with log
[ammonium] plotted versus log k (retention factor) (Figure 3-7) for the separation
of a set of -blockers, namely acebutolol, pindolol and propranolol using
ammonium acetate buffer (pH 7.5) and acetonitrile (65/35, % v/v) with a total NH4+
concentration ranging from 5 mM to 30 mM. The results revealed that when the
concentration of the competing ion increased, the retention of the investigated
analytes (positively charged under the above mentioned conditions) decreased
accordingly. This was due to the competition of ammonium ions (+ve) with the
analytes (+ve) towards the stationary phase (-ve) which resulted in the reduction of
the ion-exchange interactions of the analytes (β-blockers) with the negatively
charged stationary phase.
The ion-exchange capacity of the S1 monolithic column was determined
by an adsorption/elution method, as described in Chapter 2. The capacity
determined for a 75 µm i.d. monolithic column was 1.3 pequiv/cm of column,
corresponding to a total capacity of 15.6 pequiv for a 12 cm column. The
stationary phases exhibited relatively low cation-exchange capacity due to the
prepared monoliths having relatively large pores, decreasing the surface-to-
volume ratio, and the degree of negative charge on the polymer was low due to
the low concentration of SPMA [0.4% (w/w)] used in the polymerization mixture
[7].
Chapter 3
74
Figure 3-7: Plot of the logarithm of the retention factor for a set of β-blockers
versus the logarithm of the competing ion concentration in the mobile phase (5
to 30 mM ammonium acetate-acetonitrile 65:35, v/v), pH adjusted to 7.5 with
ammonium hydroxide, separated on S1 monolithic column [Capillary 12 cm x 75
um i.d. (capacity ≈1.3 pequiv/cm)]. Conditions: flow-rate, 0.7 µL/min. Other
conditions are the same as in Figure 3-6.
Chapter 3
75
3.3.3.2 Permeability of SPMA monolithic columns
An important characteristic of a column in LC is its permeability B0, which
represents the resistance to mobile phase flow through the column. The
permeabilities B0 for S1 and S2 monoliths were determined by pumping 30:70
acetonitrile:water (% v/v) through the column at different flow-rates and calculated
using Darcy’s Law as illustrated in Section 2.4.1. As can be seen in Figures 3-3 and
3-8, an increase in the size of both flow-through channels and their permeability
were evidenced along the 1,4-butandiol and 1-propanol variation range. Good
linearity between backpressure and the flow-rate for S1 and S2 clearly
demonstrated that the monoliths had sufficient mechanical stability to withstand
the pressure of the liquid passing through the column up to 3200 psi (Figure 3-8).
This also indicates that the prepared monolith did not appear to swell or shrink in
different flow-rates.
3.3.3.3 Method development and separation performance of SPMA
monolithic columns
Compared to the number of reports of the successful separation of large-
molecular-weight compounds using organic polymer monoliths, only a handful
have been successful for small molecules due to the lower surface areas and
generally larger pores in polymer monoliths compared to silica-based monoliths.
The present SCX-RP polymer-based monoliths are very versatile stationary phases.
With these phases, analytes with hydrophobic moieties could interact with the
hydrophobic domain of the stationary phase (RP mechanism), while positively
charged analytes could be retained by electrostatic interactions with the SCX site in
the acidic to neutral pH range.
To further characterize the selectivity of the mixed-mode poly(SPMA-co-
BMA-co-EDMA) monolithic columns under investigation, a set of acidic drugs
Chapter 3
76
Figure 3-8: Permeability measurement of different prepared monoliths in 12 cm x 75 µm i.d. capillary using acetonitrile:
water 30:70 [% (v/v)].
Chapter 3
77
and -blockers with different pKa values, namely diclofenac (pKa = 4), ibuprofen
(pKa = 4.4), ketoprofen (pKa = 5.94), arterenol (pKa = 8.55) and propranolol (pKa
= 9.5), were selected as model compounds for capillary HPLC. In terms of finding
acceptable separation conditions, two parameters were selected to access the
separation performance and repeatability: (i) the target analytes were separated
at acceptable resolution (i.e. all target analytes were baseline separated in the
chromatograms) and (ii) target analytes were separated under isocratic
conditions. Isocratic elution was preferred for simple samples (i.e. less than 10
components) where the retention factor of last peak eluted at less than 5 [34].
A mobile phase composition of 30:70 acetonitrile:water [% (v/v)]
containing 0.1% (v/v) trifluoroacetic acid (TFA) (pH 2) was selected based on the
separation mechanism (SCX-RP) of the prepared monoliths and properties of the
target analytes. Traditionally, TFA is used in the mobile phases for RP-HPLC
separations to serve one or more of the following functions: (i) pH control, (ii)
complexation with oppositely charged ionic groups to enhance RP retention (ion
pairing), (iii) or the suppression of adverse ionic interactions between basic
analytes and silanol groups on the silica to minimize peak broadening [35]. The
presence of TFA as a very strong eluting additive might have affected the elution
of the analytes. However, in a separate experiment, weaker acids, namely formic
acid and acetic acids, were used as additives [0.1% (w/w)] in the mobile phase
composition consisting of acetonitrile: water 30:70 (% v/v) for the separation of
the investigated compounds. The best analytical performance was obtained when
TFA was used as an additive in the mobile phase composition.
S1 monolithic column exhibited acceptable separation performance for the
baseline separation of five pharmaceutical drugs (Figure 3-9). At pH 2 (pH of the
used mobile phase), these β-blockers were positively charged while the acidic
Chapter 3
78
0 5 10 15 20 25 30 35 40
-6
-4
-2
0
2
0 5 10 15 20 25 30 35 40
-30
-25
-20
0 5 10 15 20 25 30 35 40
-2
0
2
4
6
8
10
0 10 20 30 40
0
5
10
23
4,5
5
4
1
5
1,2
3,4
Backpressure: 420psi
S7
Backpressure: 85psi
S1 S6Backpressure: 475psi
S2Backpressure: 195psi
2
3
5
4
1-3A
Ab
so
rbace
(m
AU
)
Time (min)
Figure 3-9: Separation of some acidic drugs and β-blockers using SPMA monolithic columns (12 cm x 75 µm i.d.)
Conditions: flow-rate, 0.5 µL/min; Peaks: 1, Arterenol; 2, ketoprofen; 3, ibuprofen; 4, diclofenac; 5, propranolol. Other
conditions are the same as in Figure 3-6.
Chapter 3
79
drugs were neutral. In terms of properties of the investigated acidic drugs, they
appeared to be hydrophobic in nature and their hydrophobicity increased from
ketoprofen, ibuprofen, to the strongly retained diclofenac. In addition, an
electrostatic attraction between the functional sulfopropyl anion of the monolith
and the positively charged β-blockers was expected. Therefore, a mixed-mode
(i.e. SCX-RP) mechanism was involved in the separation of β-blockers. Figure 3-
10 shows a fast separation on the S1 monolithic column using high flow-rate (2
µL/min) and column temperature (80 °C). The target analytes were eluted within
10 min without significantly increased backpressure. On the contrary, S2, S6 and
S7 showed poor separation of the studied compounds using the above
mentioned mobile phase composition (Figure 3-9). The surface chemistry,
concentration of sulfonic acid groups on the polymer surface as well as the size and
distribution of pores played a key role in affecting the separation performance.
3.3.3.4 Repeatability of SPMA monolithic columns
Repeatability is extremely important for a developed monolithic material
designed for the separation and sample preparation purposes. The repeatability
of the monolithic columns S1 and S2 was assessed through the %RSD of the
retention times of the five analytes mentioned above. The repeatability of the
column preparation process was tested on three levels: (i) intra-batch (run-to-run
and day-to-day) and (ii) inter-batch (batch-to-batch). The run-to-run and day to
day reproducibilities of a given monolith were evaluated from ten and six
injections, respectively, of the test analytes using the same column. Meanwhile,
the column-to-column repeatability was calculated from results obtained with six
different columns prepared from different polymerization mixtures with the
same composition. Acceptable RSD values for intra-batch and inter-batch are
2.5% and 15%, respectively, as reported in the literature [9,36,37].
Chapter 3
80
0 2 4 6 8 10 12 14
-2
-1
0
1
2
3
4
Column pressure:150psi
S1
Propranolol
Diclofenac
Ibuprofen
Ketoprofen
Arterenol
Ab
so
rba
ce
(m
AU
)
Time (min)
S1Backpressure: 150psi
Figure 3-10: Separation of some acidic drugs and β-blockers using S1 monolithic
column (12 cm x 75 µm i.d.). Conditions: mobile phase, acetonitrile/water (20:80,
v/v) with 0.1% TFA (v/v/v); flow-rate, 2 µL/min; column temperature, 80 °C.
Other conditions same as in Figure 3-6.
Chapter 3
81
The observed run-to-run (n=10) and day-to-day (n=6) RSD values of
arterenol, ketoprofen, ibuprofen, diclofenac and propranolol on S1 and S2
columns were very low, ranging from 0.5 to 2.14%, except for arterenol which
was not repeatable on the S2 column (Table 3.3). Additionally, the column-to-
column (n=6) repeatability for the aforementioned analytes on S1 and S2 columns
exhibited high RSD values, ranging from 28 to 43% (Table 3.3). Although
columns S1 and S2 exhibited good permeability (i.e. they can withstand up to
3200 psi), these results clearly confirmed the non-robustness of the prepared
monolithic columns S1 and S2 as their performance appeared to vary greatly
based on the RSD values of column-to-column repeatability of the target analytes
(Table 3.3 and Figure 3-11).
3.3.4 Effect of cross-linker on the modified SPMA monoliths(SM)
3.3.4.1 Combined effects of percentage variations of the cross-linker
and porogenic solvents on modified SPMA monoliths
The lack of column-to-column repeatability of the S1 and S2 columns was the
motivation to alter the amount of cross-linker, which was the second variable in
the studied monolithic system. A higher amount of cross-linker (EDMA) over
functional monomers (SPMA and BMA) in the fixed polymerization mixture
[40% (w/w)] monomers with respect to porogenic solvents [60% (w/w)] may
result in more robust and repeatable monolithic polymers while maintaining the
SCX-RP interaction properties. It has been reported that a higher cross-linker
concentration produced monoliths with improved column-to-column
repeatability and better mechanical stability [28]. Similar effects were observed in
the investigated system, when further experimentation was done with the same
overall monomer percentage but the monomer composition percentages being
switched between the functional monomer BMA and cross-linker EDMA.
Chapter 3
82
Table 3.3: Repeatability of prepared SPMA-based monolithic columns S1 and S2 expressed as relative standard
deviations (%RSD) of retention time (R.T.) for the separation of acidic drugs and -blockers using mobile phase 30:70
acetonitrile:water with 0.1% TFA. Other conditions are the same as in Figure 3-9.
NA – Analyte was not observed
Run-to-run (n=10) Day-to-Day (n=6) Column-to-column (n=6)
Compound
Analyte
Conc.
(mg/L)
Retention time (R.T.) Retention time (R.T.) Retention time (R.T.)
Mean %RSD Mean %RSD Mean %RSD
S1 S2 S1 S2 S1 S2 S1 S2 S1 S2 S1 S2
Arterenol 3.50 6.76 NA 1.02 NA 6.76 NA 1.39 NA 6.27 NA 17.72 NA
Ketoprofen 0.70 7.32 7.10 1.05 0.66 7.32 7.14 1.40 1.26 7.24 7.33 43.30 28.14
Ibuprofen 1.75 8.51 9.83 1.05 0.48 8.52 9.87 1.40 1.54 9.76 9.70 37.49 28.04
Diclofenac 2.45 9.50 11.51 0.98 0.87 9.50 11.44 1.27 1.25 11.57 NA 38.40 NA
Propranolol 3.50 16.55 13.50 0.83 0.74 16.51 13.36 1.35 2.14 NA 11.48 NA 36.50
Chapter 3
83
0 10 20 30 40
-2
0
2
4
6
8
10
0 10 20 30 40
-2
0
2
4
0 5 10 15 20 25 30 35 40
-2
0
2
4
6
8
0 5 10 15 20 25 30 35 40
-2
0
2
4
Ab
so
rba
ce
(m
AU
)
Time (min)
column pressure: 240psi
column pressure: 85psi
S1B
S1 C
S1A
4,532
1
5
4
3
2
1
column pressure: 275psi1
2 3
4 5
column pressure: 500psi
S1 D
1
2
3,4 5
S1ABackpressure: 85psi
S1BBackpressure: 240psi
S1CBackpressure: 275psi
S1DBackpressure: 500psi
Figure 3-11: Column-to-column repeatability of S1 (12 cm x 75 µm i.d.): separation of acidic drugs and β-blockers on
different batches of S1 stationary phase. Other conditions are the same as in Figure 3-8.
Chapter 3
84
Thus, newly modified monoliths i.e. S1M-S3M were prepared using 16% (w/w)
BUMA, 0.4% (w/w) SPMA, 23.6% (w/w) EDMA [overall 40% (w/w)] compared
with 23.6% (w/w) BUMA, 0.4% (w/w) SPMA, 16% (w/w) EDMA [overall 40%
(w/w), as for the previously prepared S1-S7] and 60% (w/w) porogenic solvents
as used previously for S1-S7 (Table 3.4).
Considering the effect of 1-propanol on the pore size shown previously in
the examples S1-S7 (where higher percentage of 1-propanol led to the formation
of monoliths with smaller pore size), the effect of increasing the amount of cross-
linker over functional monomer might be reduced if the percentage of 1-
propanol relative to that of 1,4-butandiol was varied in the preparation of the
modified monoliths S1M-S3M. Thus, fixing the amount of water [10% (w/w)], the
effect of the percentage variation of 1-propanol relative to that of 1,4-butandiol in
the 60% (w/w) porogenic solvent and 40% (w/w) monomers compositions (16%
(w/w) BMA, 0.4% (w/w) SPMA, 23.6% (w/w) EDMA) was studied in an attempt
to prepare a second set of columns, namely S1M-S3M, that were intended to
exhibit both rigidity and larger pore size.
3.3.4.2 Mercury intrusion porosimetry of the modified SPMA
monoliths
Mercury intrusion porosimetry was used to measure the pore size of the
modified SM monoliths that were prepared. The results (Table 3.4) show that the
median pore diameters from all the above prepared columns (S1M-S3M) were
2.85 µm for S2M, 1.35 µm for S1M and 1.12 µm for S3M with porogenic solvents
consisting of 1,4-butandiol, 1-propanol and water in the ratios of 20:70:10,
30:60:10 and 10:80:10 [% (w/w/w)], respectively. The pore diameters of the
modified monoliths were not what were expected. In contrast with S1-S7, there
was a higher amount of cross-linker than functional monomer in the
Chapter 3
85
polymerization mixture. As the amount of 1-propanol increased relative to that
of 1,4-butandiol, the pore size increased from S1M to S2M, although it decreased
again in S3M (Table 3.4 and Figure 3-12). This indicated that phase separation
occurred early for systems with 60 and 70% (w/w) of 1-propanol over 1,4-
butandiol and with higher amount of cross-linker over functional monomer.
3.3.4.3 Scanning electron microscopy of the modified SPMA
monoliths
In terms of the morphology of the prepared monoliths, SEM analysis of S1M, S2M
and S3M showed the copolymerized monolith was composed of a heterogeneous
surface of spherical units agglomerated into larger clusters interdispersed by large
pore channels. Therefore, these monoliths (S1M-S3M) showed good permeability
to solvents and can withstand backpressure of up to 3200 psi (Figure 3-8). It was
interesting to see that previously the S3 was not permeable, but, it became
permeable in the S3M form and macropores were observed after the composition
of the cross-linker and functional monomer were switched (Figures 3-5 and 3-13).
3.3.4.4 Separation performance, repeatability and stability of
modified SPMA monolithic columns
A similar set of acidic drugs and -blockers were tested for their
chromatographic separation on the studied monoliths (S1M-S3M). All acidic
drugs and -blockers studied in this investigation were baseline separated within
25 min using a mobile phase consisting of acetonitrile:water:TFA (34:66:0.1%,
v/v/v) for S2M and S3M columns [Figures 3-14 (a) and 3-15 (a)]. Fast separations
using S2M and S3M monolithic columns were performed using high flow-rate (2
µL/min) and column temperature (80 °C). The target analytes were eluted within
5 min [Figures 3-14 (b) and 3-15 (b)].
Chapter 3
86
Table 3.4: Effect of variation of the percentage of porogen solvents on the pore
size of the studied modified SPMA monoliths (S1M-S3M) prepared from a
polymerization mixture consisting of 0.4% (w/w) SPMA, 16% (w/w) BMA and
23.6% (w/w) EDMA that constituted 40% (w/w) of the polymerization mixture
with respect to 60 % (w/w) porogen solvents.
Polymer 1,4-butanediol 1-propanol Water Pore size
[% (w/w)] [% (w/w)] [% (w/w)] [µm]
S1M 30 60 10 1.35
S2M 20 70 10 2.85
S3M 10 80 10 1.12
Figure 3-12: Pore size distribution curve of S1M, S2M and S3M measured by
mercury intrusion porosimeter.
Chapter 3
87
(a) (b) (c)
Figure 3-13: SEM images of modified SPMA monoliths (a) S1M, (b) S2M and (c) S3M.
Chapter 3
88
0 10 20 30 40
-1
0
1
2
3
4
5
6
Ibuprofen Ketoprofen
Ab
so
rba
ce
(m
AU
)
Time (min)
Propranolol
Diclofenac
Arterenol
S2M
Column pressure:230psi
S2MBackpressure: 230psi
(a)
0 1 2 3 4 5 6 7 8
-1
0
1
2
3
4
5
Propanolol
Diclofenac
Ibuprofen
Ketoprofen
Arterenol
Ab
so
rba
ce
(m
AU
)
Time (min)
S2MColumn pressure:1025psi
S2MBackpressure: 1025psi
(b)
Figure 3-14: Separation of some acidic drugs and β-blockers on S2M monolithic
column (12 mm x 75 µm i.d.) using either mobile phase composition
acetonitrile:water:TFA (a) 34:66:0.1% v/v/v; flow-rate, 0.5 µL/min; column
temperature, 40 °C or (b) 30:70:0.1% v/v/v; 2µL/min; 80 °C. Other conditions are
the same as in Figure 3-9.
Chapter 3
89
0 10 20 30 40
0
2
4
6
8
Ibuprofen Ketoprofen
Ab
so
rba
ce
(m
AU
)
Time (min)
S3M
column pressure: 1430psi
Propranolol
Diclofenac
Arterenol
S3MBackpressure: 1430psi
(a)
0 1 2 3 4 5 6 7 8
-1
0
1
2
3
4
5
Propanolol
Diclofenac
Ibuprofen
Ketoprofen
Arterenol
Ab
so
rba
ce
(m
AU
)
Time (min)
S3MColumn pressure:3300psi
S3MBackpressure: 3300psi
(b)
Figure 3-15: Separation of some acidic drugs and β-blockers on S3M monolithic
column (12 mm x 75 µm i.d.) using either mobile phase composition
acetonitrile:water:TFA (a) 34:66:0.1% v/v/v; flow-rate, 0.5 µL/min; column
temperature, 40 °C or (b) 30:70:0.1% v/v/v; 2 µL/min; 80 °C. Other conditions are
the same as in Figure 3-9.
Chapter 3
90
Stability of a column and repeatability of retention times during
separations are critical in the evaluation of chromatographic properties. Several
batches (A-D) of S2M and S3M columns were prepared from separate
polymerization mixtures under similar conditions and the separations of the test
compounds were evaluated (Figures 3-16 and 3-17). The observed run-to-run
(n=10) RSD values based on retention times of arterenol, ketoprofen, ibuprofen,
diclofenac and propranolol on S2M and S3M columns were all within 1.2%
(Table 3.5). The RSD values for day-to-day (n=6) were higher, ranging from 0.7 to
2.7% (Table 3.5). Additionally, the column-to-column (n=6) repeatability for the
aforementioned analytes on S2M exhibited satisfactory RSD values, ranging from
3 to 11% (Table 3.5). The column-to-column (n=6) RSD values of S3M were also
at acceptable level with all values < 15% except for propranolol (16%) (Table 3.5).
The stability of the prepared S2M was impressive and acceptable separation
performance of all the analytes was obtained for up to 450 injection cycles at
80 °C (Figure 3-17). Stable retention times of tested compounds featuring a RSD
less than 3.5% were observed, even after 450 separations for the S2M monolithic
column prepared via photopolymerization. These results clearly confirmed the
robustness of the prepared monolithic column S2M as the separation
performance did not appear to deteriorate with time, temperature, number of
injections or column-to-column conditions (Figure 3-18).
3.4 Conclusions
Mixed-mode SPMA-based monolithic columns for HPLC separation of some acidic
drugs and -blockers were prepared. The influence of porogenic solvent
composition and cross-linker on morphological, porogenic and separation
properties of monoliths has been investigated. The possibility of fine-tuning of
Chapter 3
91
0 5 10 15 20 25 30 35 40 45
0
2
4
6
0 5 10 15 20 25 30 35 40 45
0
2
4
6
0 5 10 15 20 25 30 35 40 45
0
2
4
6
5
S2M batch 1
Ab
so
rbace (
mA
U)
5
S2M batch 3
S2M batch 2
Time (min)
column pressure: 250psi
column pressure: 235psi
4
321
432
1
5column pressure: 392psi
1
23
4
S2M batch 2Backpressure : 250psi
S2M batch 3Backpressure : 392psi
S2M batch 1 Backpressure: 235psi
Time (min)
Figure 3-16: Column-to-column repeatability of S2M: separation of acidic drugs and β-blockers on different batches of
S2M stationary phase using mobile phase composition acetonitrile:water:TFA (34:66:0.1%, v/v/v). Other conditions are
the same as in Figure 3-8.
Chapter 3
92
0 5 10 15 20 25 30 35 40 45
0
2
4
6
0 5 10 15 20 25 30 35 40 45
0
2
4
6
0 5 10 15 20 25 30 35 40 45
-2
0
2
4
6
5
S3M batch C1
Ab
so
rbace (
mA
U)
5
S3M batch C3
S3M batch C2
Time (min)
column pressure: 1660psi
column pressure: 1360psi
43
2
1
43
2
1
5
column pressure: 930psi1
23
4
Time (min)
S3M batch 2Backpressure: 1660psi
S3M batch 3Backpressure: 930psi
S3M batch 1 Backpressure: 1360psi
Figure 3-17: Column-to-column repeatability of S3M: separation of acidic drugs and β-blockers on different batches of
S3M stationary phase using mobile phase composition acetonitrile:water:TFA (34:66:0.1%, v/v/v). Other conditions are
the same as in Figure 3-9.
Chapter 3
93
Table 3.5: Repeatability of prepared SPMA-based modified monolithic columns S2M and S3M expressed as relative
standard deviations (%RSD) of retention time (R.T.) for the separation of acidic drugs and -blockers. Other conditions
are the same as in Figure 3-16.
Run-to-run (n=10) Day-to-Day (n=6) Column-to-column (n=6)
Compounds
Analyte
Conc.
(mg/L)
Retention time (R.T.) Retention time (R.T.) Retention time (R.T.)
Mean %RSD Mean %RSD Mean %RSD
S2M S3M S2M S3M S2M S3M S2M S3M S2M S3M S2M S3M
Arterenol 3.50 5.92 6.56 0.72 0.82 5.92 6.52 0.74 0.87 6.01 7.16 3.45 6.19
Ketoprofen 0.70 7.39 8.42 0.76 0.88 7.38 8.36 1.10 0.89 7.66 9.30 4.90 2.96
Ibuprofen 1.75 9.17 10.71 0.84 0.95 9.16 10.66 1.14 1.38 9.97 12.09 5.08 5.44
Diclofenac 2.45 11.30 13.31 0.92 0.98 11.29 13.27 1.38 1.95 12.73 15.31 5.03 8.85
Propranolol 3.50 13.61 16.92 0.82 1.17 13.60 16.64 1.49 2.72 15.95 21.58 11.05 15.97
Chapter 3
94
Re
ten
tio
n t
ime
(R
.T.)
Arterenol
Ketoprofen
Ibuprofen
Diclofenac
Propranolol
Figure 3-18: Study of the stability of S2M column after 450 injection cycles of a mixture of acidic drugs and -blockers.
Conditions: mobile phase, acetonitrile:water (30:70, v/v) with 0.1% TFA; flow-rate, 2 µL/min; 1 µL injection volume;
column temp, 80 °C.
Chapter 3
95
porosity and separation properties of monoliths was explored by changing the 1-
propanol to 1,4-butanediol ratio to allow for the optimization of the HPLC
performance of the columns. In general, at a given porogenic solvent composition,
a SPMA-based monolith with a greater amount of cross-linker gave better
repeatability values in separation behavior, even at high temperature (up to 80 °C).
Furthermore, the separation mechanism of the investigated compounds appeared
to be based on a mixed-mode hydrophobic/ion-exchange interaction of the analytes
with the SCX-RP monolith. The influence of ion-exchange interaction on the
retention between the -blockers and the stationary phase could be manipulated
by changing the concentration of the competing ion (5-30 mM NH4+) of the mobile
phase used. All these properties are promising for a broader application of mixed-
mode porous polymer monoliths in low resolution chromatography and sample
preparation for pharmaceutical analysis. The simplicity of the in situ preparation
process also makes the monolithic columns excellent candidates for use in
miniaturized devices, such as microfluidic chips and pipette tips.
3.5 References
[1] R.S. Plumb, W.B. Potts, 3rd, P.D. Rainville, P.G. Alden, D.H. Shave, G.
Baynham, J.R. Mazzeo, Rapid Commun. Mass Spectrom. 22 (2008) 2139.
[2] H. Toll, R. Wintringer, U. Schweiger-Hufnagel, C.G. Huber, J. Sep. Sci. 28
(2005) 1666.
[3] J. Zhang, S.L. Wu, J. Kim, B.L. Karger, J. Chromatogr. A 1154 (2007) 295.
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Chromatogr. A 1216 (2009) 6824.
[5] Y. Hsieh, Expert Opin. Drug Metab. Toxicol. 4 (2008) 93.
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[7] J.P. Hutchinson, E.F. Hilder, R.A. Shellie, J.A. Smith, P.R. Haddad, Analyst
131 (2006) 215.
[8] J. Liu, I. White, D.L. DeVoe, Anal. Chem. 83 (2011) 2119.
[9] L. Geiser, S. Eeltink, F. Svec, J.M. Frechet, J. Chromatogr. A 1140 (2007) 140.
[10] H. Minakuchi, K. Nakanishi, N. Soga, N. Ishizuka, N. Tanaka, Anal. Chem.
68 (1996) 3498.
[11] E.F. Hilder, F. Svec, J.M. Frechet, J. Chromatogr. A 1044 (2004) 3.
[12] F. Kilar, S. Hjerten, J. Chromatogr. 480 (1989) 351.
[13] Q.C. Wang, K. Hosoya, F. Svec, J.M. Frechet, Anal. Chem. 64 (1992) 1232.
[14] F. Svec, J. Chromatogr. A 1217 (2010) 902.
[15] G. Guiochon, J. Chromatogr. A 1168 (2007) 101.
[16] K.C. Saunders, A. Ghanem, W. Boon Hon, E.F. Hilder, P.R. Haddad, Anal.
Chim. Acta 652 (2009) 22.
[17] Z. Jiang, N.W. Smith, P.D. Ferguson, M.R. Taylor, J. Sep. Sci. 31 (2008) 2774.
[18] B. Gu, Z. Chen, C.D. Thulin, M.L. Lee, Anal. Chem. 78 (2006) 3509.
[19] Z. Liu, R. Wu, H. Zou, Electrophoresis 23 (2002) 3954.
[20] W. Wieder, C.P. Bisjak, C.W. Huck, R. Bakry, G.K. Bonn, J. Sep. Sci. 29 (2006)
2478.
[21] H. Fu, C. Xie, J. Dong, X. Huang, H. Zou, Anal. Chem. 76 (2004) 4866.
[22] J. Lin, J. Lin, X. Lin, Z. Xie, J. Chromatogr. A 1216 (2009) 801.
[23] X. Wang, K. Ding, C. Yang, X. Lin, H. Lu, X. Wu, Z. Xie, Electrophoresis 31
(2010) 2997.
[24] H. Fu, C. Xie, H. Xiao, J. Dong, J. Hu, H. Zou, J. Chromatogr. A 1044 (2004)
237.
[25] M. Bedair, Z. El Rassi, J. Chromatogr. A 1013 (2003) 35.
[26] C. Bisjak, R. Bakry, C. Huck, G. Bonn, Chromatographia 62 (2005) s31.
Chapter 3
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[27] C. Gatschelhofer, C. Magnes, T.R. Pieber, M.R. Buchmeiser, F.M. Sinner, J.
Chromatogr. A 1090 (2005) 81.
[28] Y. Li, H.D. Tolley, M.L. Lee, J. Chromatogr. A 1217 (2010) 4934.
[29] D. Schaller, E.F. Hilder, P.R. Haddad, Anal. Chim. Acta 556 (2006) 104.
[30] D.S. Peterson, T. Rohr, F. Svec, J.M. Frechet, Anal. Chem. 75 (2003) 5328.
[31] S. Xie, F. Svec, J.M.J. Fréchet, J. Polym. Sci. Part A: Polym. Chem. 35 (1997)
1013.
[32] X. Huang, Q. Wang, H. Yan, Y. Huang, B. Huang, J. Chromatogr. A 1062
(2005) 183.
[33] E.F. Hilder, F. Svec, J.M. Frechet, Electrophoresis 23 (2002) 3934.
[34] A.P. Schellinger, P.W. Carr, J. Chromatogr. A 1109 (2006) 253.
[35] Y. Chen, A.R. Mehok, C.T. Mant, R.S. Hodges, J. Chromatogr. A 1043 (2004)
9.
[36] J. Lin, G. Huang, X. Lin, Z. Xie, Electrophoresis 29 (2008) 4055.
[37] A. Palm, M.V. Novotny, Anal. Chem. 69 (1997) 4499.
Chapter 4
98
C h a p t e r 4
Development of a Trypsin-Immobilized
Monolithic Polymer with Pipette-Tip Format
for Protein Digestion
4.1 Introduction
There have been significant improvements in the analytical methodologies and
techniques pertaining to proteomic studies in the past decades. Several strategies
have emerged as efficient and indispensable techniques for the bottom-up
approach of protein analysis, which involves separation, identification, and
characterization of proteins in the field of functional proteomics [1,2]. Two-
dimensional electrophoresis (2-DE) is one of the most powerful methods for the
separation of proteins, followed by off-line digestion and further identification by
MS/MS [3]. Alternatively, the hyphenation of multidimensional HPLC with
MS/MS is used for the separation and identification of all the digested proteins
extracted from a biological sample [4]. However, sample preparation often poses a
constraint to rapid bioanalysis. The traditional protocol for protein digestion is
accomplished by enzymatic hydrolysis in free solution for at least several hours.
This method presents a number of drawbacks, such as proteolytic enzyme
autodigestion, low efficiency, extended incubation time, insufficient sample
loading capacity for direct analysis of plasma, and manual sample manipulation
steps. These limit the advancement of high throughput protein identification
technology [5]. To obtain rapid, sensitive and high-throughput protein digestion,
research efforts have focused on the immobilization of enzymes.
Chapter 4
99
Research dealing with immobilization of trypsin can be dated back to the
late 1970s [6]. Immobilized enzymes have found applications in a wide variety of
areas. In addition, the application of bioreactors containing immobilized enzymes
is also growing as an integral part of quality control (QC) and quality assurance
(QA) for products in biotechnology, chemical synthesis, and the pharmaceutical
industry [7]. These enzyme reactors enable rapid screening of enzyme inhibitor
candidates as well as detailed characterization of binding interactions and reaction
mechanism [8,9]. Over the past few decades, various strategies enabling enzyme
immobilization have been designed and developed. Enzymes can be covalently
bound, trapped, or physically adsorbed onto various supports, such as particles
[10], membranes [11], the inner walls of fused-silica capillaries,[12] magnetic
particles[13] and monolithic materials [14]. It is also now well-known that the
properties of the support such as pore size, porosity, chemistry, and mechanical
strength will affect the characteristics of the immobilized enzymes [15]. That is, the
activity and the applicability of the resulting bioreactor are greatly affected by the
selection of the support matrix.
Currently, monoliths have been widely used as matrices for sample
preparation and separation. The micrometer-sized pores of the monoliths provide
a low-pressure drop for the proteins to flow convectively through the interstices
and interact with the enzyme-immobilized surfaces via short diffusion distances,
which may enhance the digestion efficiency. A review of the advantages of using
monoliths over using alternative phases for bioanalytical applications has been
reported elsewhere [16]. In addition, the wide variety of applications of silica and
organic polymer monolithic phases in the preparation of immobilized enzyme
reactors of different formats has been reported. A number of bioreactors prepared
by inorganic silica monoliths in 4.6 mm i.d. column have been studied by
Chapter 4
100
Temporini et al. for the on-line digestion and characterization of proteins [17,18]. A
solid-phase digestion tool of immobilized-trypsin on monolithic silica gel in a
pipette tip has been developed [19]. Organic polymer monoliths used as supports
for enzyme immobilization have been prepared in different formats, such as in situ
in the channel of microfluidic devices [14], monolithic disks [20] and columns of
different i.d. [7,21]. Specifically, organic polymer monoliths have gained much
attention over silica-based monoliths as supports for enzyme immobilization due
to properties such as high chemical stability over a wide pH range, good
biocompatibility, ease of preparation in different formats and ease of modification
with various functional groups [5].
A further advantage of polymer monoliths is that they can be readily
modified, for example using UV light. Irradiation with UV light through a
photomask allows precise patterning of the area subjected to surface modification
and enables the precise placement of specific functionalities within selected areas of
a single monolith for use in a variety of applications [22,23]. This process permits
the introduction of multiple sites with various functionalities located next to each
other or at predetermined locations in a single monolith [24]. Recently, 2-vinyl-
4,4,-dimethyazlactone (VAL) has been used for copolymerization with ethylene
glycol dimethacrylate (EDMA) to obtain a reactive monolithic support for the
immobilization of enzymes [25,26]. Krenkova et al. [7] described the preparation of
enzymatic microreactors containing trypsin and endoproteinase LysC immobilized
on a porous polymer monolith for the characterization and identification of
proteins, such as cytochrome c, bovine plasma albumin, and high-molecular
weight human immunoglobulin G. Subsequently, a reactor with immobilized
peptide-N-glycosidase F on a porous polymer monolith in a capillary was also
developed for the fast and efficient release of N-linked glycans from
Chapter 4
101
immunoglobulin G molecules [7]. The monolith was first hydrophilized via
photografting of poly(ethylene glycol) methacrylate (PEGMA) followed by
photografting of VAL. This multistep photografting process was used to reduce
non-specific adsorption of proteins and to obtain a support containing reactive
azlactone functionalities so as to enable the preparation of highly active
immobilized enzymes [7,26]. With the use of this system, a much shorter reaction
time and a lower reaction temperature were achieved for the protein digestion.
In this chapter, a novel trypsin-immobilized organic polymer monolith
based on a pipette tip format is described for the analysis of proteins. A robust and
reproducible monolith formulation reported previously in Chapter 3 was used as
the support for the enzyme immobilization. The tips were prepared using a porous
polymer monolith, followed by photografting the pore surface of the monolith
with VAL inside the pipette tip. An unstable enzyme, trypsin, was successfully
immobilized covalently in the azlactone-functionalized area. The functionalized
monolith was characterized, followed by evaluation of its efficiency in the rapid
digestion of proteins.
4.2 Experimental
The general experimental details are described in Chapter 2. Detailed conditions
are elaborated in each of the figure captions.
4.2.1 Fluorescent assay of protein absorption
4.2.1.1 Photografting of poly(ethylene glycol) methacrylate (PEGMA)
A two-step modification reaction was used for the hydrophilization of the
monolithic support to prevent the non-specific adsorption of proteins to the
monolith surface [26]. Briefly, the monolith in the pipette tip was flushed with a
Chapter 4
102
deaerated 5% (w/w) benzophenone (BP) solution in methanol for 30 min using a
vacuum filtration apparatus. It was then exposed to UV irradiation for 4 min with
rotation. The tip was fitted on a rotating stirrer and was rotated at the slowest
speed possible (~50 rpm). After photografting BP, the monolith was flushed with
methanol for 30 min to remove the unbound initiator. Next, a deaerated 0.1 M
solution of PEGMA monomer in water was pumped through the monolith for 30
min and the monolith was exposed to UV irradiation for 4 min with rotation. The
monolith was then rinsed with water for 60 min to remove the unreacted PEGMA
monomer.
4.2.1.2 Preparing stock solution of protein and fluorescein
isothiocyanate (FITC)
A stock solution of protein was prepared by directly dissolving commercially
available bovine serum albumin (BSA, Sigma Aldrich) in MilliQ water. In order
to prepare 1x10-4 M of BSA (MW 66430), 132.9 mg of BSA was dissolved in 20 mL
of water using a magnetic stirrer at approximately 25 °C. Then, it was diluted to
1x10-5 M with water. To prepare 10-3 M of FITC (Sigma Aldrich) solution, 19.5 mg
of FITC was dissolved in 50 mL of high purity (>99.8%) acetone and diluted to
5x10-5 M with acetone.
4.2.1.3 Labeling reaction of protein with fluorescein isothiocyanate
(FITC-BSA)
In order to prepare a fluorescently-labeled BSA solution with the concentration
of 5x10-6 M, 20 mL of BSA solution (1x10-5 M) and 20 mL FITC (5x10-5 M) were
mixed (five moles of FITC were coupled to one mole of albumin) in dry 50 mL
plastic tubes. Then the mixtures were wrapped with aluminum foil and kept in
the drawer overnight to perform the labeling reaction (at ~20-25 °C temperature
controlled environment).
Chapter 4
103
4.2.1.4 Fluorescent assay of protein adsorption on the pipette tips
The extent of protein adsorption on unmodified and PEGMA-photografted
monolith in situ pipette tips was evaluated using a fluorescence assay developed
previously [27]. Briefly, the pipette tips were flushed with a 5x10-6 M (~0.33
mg/mL) solution of fluorescein-labeled BSA for 1 h. The pipette tips were then
rinsed with water for 30 min to remove excess BSA. After BSA exposure, the
cross-section of the tip was then observed using a microscope with a blue LED
array illuminating the microscope for fluorescent imaging as shown in Figure 4-1.
Monolith tips with and without PEGMA-photografting were used for screening
non-specific protein adsorption. Successful prevention of protein adsorption was
indicated by the low fluorescence intensity observed. By contrast, high
fluorescence intensity indicated substantial absorption of the fluorescent protein
and poor performance of the monolith surface.
Figure 4-1: The set-up of a microscope with blue LED array illuminating the
microscope for fluorescent imaging.
Chapter 4
104
4.2.2 Photografting of 2-vinyl-4,4-dimethylazlactone (VAL)
A mixture consisting of 25% (w/w) VAL and 0.22% (w/w) BP dissolved in 75:25 (%
w/w) tert-butyl alcohol:water was pumped through the monolith for 30 min and
exposed to UV irradiation for 30 min on both sides of the tips (Figure 4-2). After
grafting vinyl azlactone, the monolith was washed with acetone for 1 h to remove
excess reagents.
4.2.3 Immobilization of trypsin on grafted support
Trypsin (1, 2, 5, 10 and 15 mg/mL) was dissolved separately in 50 mM phosphate
buffer, pH 7.2, containing 0.25 mg/mL benzamidine. Benzamidine was added to
avoid undesirable autodigestion and the enzyme solution was pipetted for 15 to 20
cycles. After the immobilization reaction, the monoliths were rinsed with 1 M
ethanolamine in 50 mM phosphate buffer, pH 7.2 to quench all unreacted azlactone
functionalities. Finally, the monolithic reactor was washed and stored with 50 mM
ammonium acetate solution pH 6.7 at 4 C.
The amount of immobilized trypsin on the azlactone monolith in the pipette
tips was determined by the modified method described previously [28]. After the
immobilization process, the tip was rinsed with 20 μL of the same buffer to remove
the unreacted trypsin on the surface of monolith. This eluent was collected and
combined with the eluent from the immobilization and diluted to 2 mL and the
absorbance was measured at 280 nm. The concentration was calculated by the
difference of the amount of trypsin before and after immobilization using the
calibration curve established (slope = 0.0014 abs/µgmL-1).
4.2.4 Sample preparation for protein digestion
The sample preparation for protein digestion was performed according to the
procedure described previously [7]. The digestion of proteins using immobilized
Chapter 4
105
Figure 4-2: Schematic diagram of the photopatterning process. (a) vinylazlactone (VAL) is photopatterned onto the
monolith surface and activates the surface for protein immobilization. (b) azlactone functionality reacts with amines of
proteins to form a covalent amide bond between the protein and the polymer monolith surface.
Chapter 4
106
enzyme polypropylene pipette (IMEPP) tips is illustrated in Figure 4-3. Briefly,
cytochrome c was dissolved in 50 mM ammonium acetate, pH 8.75 containing 20%
acetonitrile to a concentration of 0.5 mg/mL. 100 µL of the protein sample was
incubated in a dry-bath incubator for 5 min at 37 C before being pipetted with
IMEPP tips using the protocol described in Section 2.3.9. For protein digestion with
soluble enzyme, trypsin was added at a substrate-to-enzyme ratio of 50:1 (w/w) for
protein digestion and the solution was incubated at 37 C for 24 h. The proteolysis
was terminated by decreasing the pH with the addition of formic acid to a final
concentration of 0.1% (1 µL of 10% formic acid in water). The resultant peptide
fragments from the digestion of IMEPP tips or liquid phase digestion were
collected in microvials and analyzed using direct infusion MS. The mass spectra of
all samples were then compared against a protein digestion database (protein
prospector, http:// prospector.ucsf.edu/) to identify the digest and determine the
sequence coverage (Figure 4-3).
4.3 Results and discussion
4.3.1 Surface modification of the polypropylene (PP) tips
The modification of the surface of PP was first demonstrated by Stachowiak et al.,
who covalently attached the porous monolithic polymer to the tip wall in order to
transfer the technique to the microfluidic chips [22,29]. Following this, Altun et al.
have used the grafting modification to create reactive groups and they improved
the wetting ability of the inner surfaces of PP tips in order to prepare monolithic
pipette tips for SPE purposes in high throughput bioanalysis [29,30]. In the present
work, the inner surface of PP tips was modified via grafting of monomer, followed
by the preparation of a porous poly (butyl methacrylate-co-ethylene glycol
dimethacrylate) monolith. The porous polymer monolith in situ PP tips can act as a
Chapter 4
107
Figure 4-3: Schematic diagram for the protein digestions using trypsin-immobilized monolith tip.
Chapter 4
108
support for enzyme immobilization in protein digestion. In this study, we also
investigated a two-step photografting approach that involved covalent attachment
of monomer to the PP surface (method described in section 2.3.7). Figure 4-4 shows
the SEM images of non-modified PP tips and surface modification of tips by single-
and two-step photografting using BP as initiator. The SEM images were not
markedly different except that the surface of the modified tip looked smoother, as
shown in Figures 4-4 (b) and 4-4 (c).
Based on the results above, the effects of single- and two-step surface
modification were further investigated by synthesizing the poly (BMA-co-EDMA)
in situ PP tip using UV-induced polymerization. Figure 4-5 (a) shows SEM images
of the monolith inside the PP tips in the absence of surface treatment. A magnified
image shown in Figure 4-5 (a) bottom indicates a large void between the PP wall of
the tip and the monolith. After washing the monolith with methanol and drying it
in the vacuum oven, the monolith was loosened and could slip out of the tip. By
contrast, a surface grafted with monomer enabled a better attachment of the
monolith either by single-step or two-step photografting methods, as shown in
Figures 4-5 (b) and 4-5 (c). This can be confirmed by the presence of a thin layer of
polymer gel observed from the magnified images taken between the PP wall and
monolith [Figures 4-5 (b)] which can also correspond to the smoother surfaces of
tip walls for single or two-step photografting. Both of the photografting methods
showed excellent covalent binding of the monolith to the PP surface and good
mechanical stability. When a high pressure was applied, the monolith attached
well to the surface without slipping out.
In addition, the differences between using EDA (ethylene diacrylate) and
EDMA for the surface modification of PP tips were compared. Previous works
suggested that EDA offers the benefits of shorter exposure time which
Chapter 4
109
(a) (b) (c)
Figure 4-4: SEM images of PP tips (above) and the magnified part (bottom): (a) no surface modification (b) single-step
photografting and (c) two-step photografting.
Chapter 4
110
(a) (b) (c)
Figure 4-5: SEM images of porous polymer monoliths prepared in PP tips (above) and the magnified part (bottom): (a) no
surface modification (b) single-step photografting with MMA/EDMA 1:1 with BP [3% (w/w)] and (c) two-step
photografting.
Chapter 4
111
achieve a stronger surface treatment of the PP surface. However in this work,
surface modification with EDA achieved only partial attachment of the monolith to
the PP wall. These results are in agreement with findings reported by Stachowiak
et al. who stated that using EDMA for surface modification achieved the best result,
with no visible void between the PP wall and monolith [22]. Therefore, a single-
step photografting with EDMA was used to simplify the preparation of monoliths
in tips.
4.3.2 Fluorescence assay of protein adsorption
The hydrophilic polymer PEGMA has been widely used for modifying the surfaces
of monoliths to reduce non-specific protein interactions and to permit repeated use
of the reactor [7,26,31]. BSA is well-known to be a “sticky” protein and is
commonly used to estimate protein adsorption onto enzyme-immobilized
monolithic supports [32]. The fluorescence assay of BSA can be used to mimic the
conditions for enzyme immobilization onto the monolith to obtain an enzyme
reactor in pipette tip format [27]. Figure 4-6 shows the fluorescence images of a
pipette tip with and without PEGMA-grafted after being flushed with fluorescein-
labeled BSA and rinsed with water. The non-PEGMA-grafted tip was highly
susceptible to absorption of the fluorescently-labeled protein. The monolith
photografted with PEGMA [Figure 4-6 (b)] exhibited lower fluorescent intensity
than the monolith which had not been photografted [Figure 4-6 (a)]. Despite the
fact that this method was developed to impart hydrophilic functionalization onto
monoliths in small i.d. capillaries (75 µm), these images clearly demonstrated the
successful upscaling of the reaction to larger i.d. PP tips. When monolith
containing FITC-BSA was washed with 20% acetonitrile in water, most of the BSA
was removed, with much lower fluorescence intensity monolith observed [Figure
4-6 (c)]. The longer photografting time (from 4 min to 8 min) for PEGMA resulted
in a thicker layer of PEGMA being formed on the monolith, which caused high
Chapter 4
112
(a) (b) (c)
Figure 4-6: Fluorescence cross-section images of pipette tips. Fluorescein-labeled BSA was pumped through the polymer
monolith tips (a) without and (b) PEGMA-grafted region after washed with water and (c) 20:80 (% v/v) acetonitrile:water.
113
backpressure. Therefore, 4 min photografting was deemed to be the optimum
irradiation time.
4.3.3 Enzyme immobilization using azlactone functionalities
The application of azlactone monoliths in enzyme immobilization has been widely
used in the past 10 years for the fabrication of enzymatic microreactors. The aim of
this chapter was to develop an inexpensive single-use IMEEP tip, so the issue of
non-specific adsorption is less of a problem in this work. Thus, we used the non-
PEGMA-grafted monolithic PP tips for subsequent studies.
As a starting point, the protocols described previously [26] were used to
prepare the azlactone monolith [i.e. 15% (w/w) VAL, 2 min photografting and 5
mg/mL of trypsin for immobilization]. The first demonstration of proteolytic
activity for the IMEPP tip was carried out by pipetting 100 µL 0.5 mg/mL of
cytochrome c for 10 cycles. However, no noteworthy enzyme activity was
observed for the cytochrome c digestion using the IMEPP tip [Figure 4-7 (b)], as
compared to the cytochrome c solution digested using the non-VAL-grafted
monolithic PP tip [Figure 4-7 (a)]. This might be due to the fact that the UV source
used for functionalization could not penetrate the large i.d. of the monolith
fabricated in situ inside the PP tips, since the PP materials have a much lower UV
transmission compared to the UV transparent fused-silica capillaries with smaller
i.d.. Another possibility for the lack of enzyme activity might be an insufficient
amount of VAL grafted onto the surface of monolith, resulting in a low capacity of
enzyme immobilized on the polymer surface.
As demonstrated by Krenkova et al. [7], the concentration of VAL at 25%
(w/w) or above in the monolith support clogged the column. Here, 25% (w/w) VAL
was used in the photografting mixture, with 10 min of UV irradiation. The result
shows that reasonable backpressure could be maintained to allow solution to pass
Chapter 4
114
through the PP tips after photografting with the relatively high concentration of
VAL. Cytochrome c was again used to demonstrate the proteolytic activity of the
IMEPP tip grafted with higher concentration of VAL. Figure 4-7 (c) indicates that
the IMEPP exhibited very high activity for the complete digestion of cytochrome c
with 100% of sequence coverage achieved within few minutes.
The maximum concentration of VAL and photografting time that could be
used to maximize the enzyme loading capacity of the IMEPP tip were also
determined. Firstly, the photografting time was increased from 10 min to 30 min
while maintaining the permeability of the monolith. However, when the
concentration of VAL was increased from 25% (w/w) to 30% (w/w), permeability
through the IMEPP tip was no longer observed. Therefore, 25% (w/w) VAL in the
photografting mixture and 30 min photografting time were used for the following
studies. Table 4.1 shows a comparison of the performance of the IMEPP tips with
different numbers of pipetting cycles for the digestion of cytochrome c at 0.1
mg/mL. This table shows that the highly efficient IMEPP tip developed here could
give sequence coverage of 89% even with 1 pipetting cycle (or 10 s) of digestion of
low-molecular weight proteins, such as cytochrome c.
4.3.4 Effects of trypsin concentration for immobilization on
monolithic support
A batch of three IMEPP tips immobilized with different concentrations of trypsin
was prepared (2530a, b, c, d and e). Using the UV spectrometric method, the
amount of immobilized trypsin on the azlactone monolith was found to be
different when different concentrations of trypsin solution were employed (Table
4.2). The maximum bound amount of trypsin was limited to about 140 µg which
was
Chapter 4
115
453.4 679.5
1546.0
1766.6
2061.0
2248.2
+MS, 0.3-0.7min #(24-53)
0
20
40
60
80
100
Intens.
[%]
250 500 750 1000 1250 1500 1750 2000 2250 m/z (a)
129.1276.7350.7
453.4679.5
728.7817.8
971.2
1545.9
1766.5
2061.0
+MS, 0.2-0.5min #(12-34)
0
20
40
60
80
100
Intens.
[%]
250 500 750 1000 1250 1500 1750 2000 2250 m/z (b)
261.2
454.3
536.3
584.8
648.9
779.5
907.6
957.2
1168.7
1296.8
1456.4
1546.0
1766.7
1894.0
2061.0
+MS, 0.8-1.9min #(56-144)
0
20
40
60
80
100
Intens.
[%]
250 500 750 1000 1250 1500 1750 2000 2250 m/z (c)
Figure 4-7: ESI-TOF mass spectra obtained from cytochrome c digestion using
pipette tips (a) without VAL photografting; (b) photografting under UV light at
an exposure time of 2 min with 15% (w/w) VAL in photografting mixture; (c)
exposure time of 30 min with 25% (w/w) VAL in photografting mixture.
Chapter 4
116
Table 4.1: Comparison of digestion performances of the IMEPP tip with
different numbers of pipetting cycles for the digestion of cytochrome c.
Label 2530 2530 2530 2530
pipette cycles 1 5 10 15
Identified peptide
number 87/105 99/105 100/105 105/105
Sequence coverage (%) 89 94 95 100
Table 4.2: Amount of bound trypsin and performance of the IMEPP tips
immobilized with different concentrations of trypsin.
Label 2530a 2530b 2530c 2530d 2530e
Trypsin conc. immobilization
(mg/mL) 1 2 5 10 15
Reacted amount (Trypsin µg) 214 406 1031 1809 2821
Bound amount (Trypsin µg) 18 71 138 142 135
Identified peptide number 54/105 95/105 100/105 102/105 99/105
Sequence coverage (%) 51 90 95 97 94
Chapter 4
117
obtained using a concentration of 10 mg/mL trypsin in solution. Any trypsin
concentration greater than 10 mg/mL makes it difficult to pass through the
monolith because of the high backpressure stemming from the high viscosity
solution. However, the amount of immobilized enzyme on the monolith in the
present work was four times higher than that previously used in a capillary format
[28]. Thus, it might result in much higher digestion capacity.
With regard to enzyme immobilization, it is always desirable to increase the
amount of enzyme on the support in order to enhance the enzyme:substrate ratio
and thus increase the protein digestion reaction rate. Two critical factors have been
suggested for the optimization of trypsin bioreactor performance: accessibility to
the active site related to enzyme:substrate ratio, and response intensity (i.e.
sensitivity) related to the total amount of active sites [33]. As shown in Table 4.2,
when the amount of trypsin used for immobilization increased, the bound amount
of trypsin as well as the sequence coverage likewise increased. However, when the
immobilization maximum had been reached, using 10 mg/mL of trypsin, all the
available binding sites had been occupied. When 15 mg/mL of trypsin solution was
used for immobilization, the sequence coverage slightly decreased. This might
either be due to the high enzymatic density on the solid support which hindered
the access of substrate to the active site, or perhaps the higher enzyme loading
induced an aggregation and/or denaturation of the enzyme prior to or after
immobilization, which led to a slight decrease in the enzyme activity [33]. Based on
these results, a trypsin concentration of 10 mg/mL for immobilization was selected
for studies of protein digestion.
In summary, a single-step surface modification method with EDMA, post
modification of monolith support with 25% (w/w) VAL concentration and exposed
under UV for 30 min, enzyme immobilization using 10 mg/mL of trypsin and 50
Chapter 4
118
mM ammonium acetate solution in 20:80 acetonitrile:water, pH 8.75, were used in
all our experiments.
4.4 Conclusions
In this work, a novel trypsin-immobilized polypropylene pipette tip based on an
organic polymer monolith was developed. UV-initiated photografting provided a
simple and versatile approach for PP surface modification that produced better
attachment of the polymer monolith. Further post-modification on the pore surface
of the monolith created reactive azlactone functionalities which afforded an
excellent support for the immobilization of trypsin. In addition, the high surface
area to volume ratio, as well as a high degree of interconnected channels produced
in photopolymerized monoliths, provided rapid mass transfer for proteolytic
digestor applications. The preliminary results obtained are very promising in view
of the very high sequence coverage coupled with very short digestion time
achieved for cytochrome c. It is likely that this developed IMEPP tip can be used
for the digestion of protein samples of different sizes. This makes the developed
immobilized enzyme reactor based on pipette tip format very suitable for high-
throughput sample preparation required to accelerate the process of protein
mapping, drug discovery and drug development. Further studies were to be
focused on detailed characterization of this enzyme reactor in comparison with
commercially available tryptic digest tips and traditional liquid phase digestion. In
addition, the developed IMEPP tips were to be used for fast sample preparation of
biological samples of pharmaceutical interest for both qualitative and quantitative
bioanalysis.
Chapter 4
119
4.5 References
[1] K. Zhang, S. Wu, X. Tang, N.K. Kaiser, J.E. Bruce, J. Chromatogr. B Analyt.
Technol. Biomed. Life Sci. 849 (2007) 223.
[2] S. Ota, S. Miyazaki, H. Matsuoka, K. Morisato, Y. Shintani, K. Nakanishi, J.
Biochem. Biophys. Methods 70 (2007) 57.
[3] S.M. Hanash, Electrophoresis 21 (2000) 1202.
[4] M.P. Washburn, D. Wolters, J.R. Yates, Nature Biotechnology 19 (2001) 242.
[5] J. Ma, L. Zhang, Z. Liang, W. Zhang, Y. Zhang, J. Sep. Sci. 30 (2007) 3050.
[6] J. Turkova, K. Blaha, M. Malanikova, D. Vancurova, F. Svec, J. Kalal,
Biochim. Biophys. Acta Enzymol. 524 (1978) 162.
[7] J. Krenkova, N.A. Lacher, F. Svec, Anal. Chem. 81 (2009) 2004.
[8] J. Krenkova, F. Svec, J. Sep. Sci. 32 (2009) 706.
[9] S.J. Lovell, L.W. Nichol, D.J. Winzor, FEBS Lett. 40 (1974) 233.
[10] E. Bonneil, M. Mercier, K.C. Waldron, Anal. Chim. Acta 404 (2000) 29.
[11] Y. Liu, H. Lu, W. Zhong, P. Song, J. Kong, P. Yang, H.H. Girault, B. Liu,
Anal. Chem. 78 (2006) 801.
[12] A. Bossi, L. Guizzardi, M.R. D'Acunto, P.G. Righetti, Anal. Bioanal. Chem.
378 (2004) 1722.
[13] A. Le Nel, J. Krenkova, K. Kleparnik, C. Smadja, M. Taverna, J.L. Viovy, F.
Foret, Electrophoresis 29 (2008) 4944.
[14] D.S. Peterson, T. Rohr, F. Svec, J.M.J. Frechet, Anal. Chem. 74 (2002) 4081.
[15] J. Krenkova, F. Svec, J. Sep. Sci. 32 (2009) 706.
[16] K.C. Saunders, A. Ghanem, W.B. Hon, E.F. Hilder, P.R. Haddad, Anal.
Chim. Acta 652 (2009) 22.
[17] C. Temporini, E. Calleri, K. Cabrera, G. Felix, G. Massolini, J. Sep. Sci. 32
(2009) 1120.
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[18] C. Temporini, L. Dolcini, A. Abee, E. Calleri, M. Galliano, G. Caccialanza, G.
Massolini, J. Chromatogr. A 1183 (2008) 65.
[19] S. Miyazaki, K. Morisato, N. Ishizuka, H. Minakuchi, Y. Shintani, M.
Furuno, K. Nakanishi, J. Chromatogr. A 1043 (2004) 19.
[20] C. Delattre, P. Michaud, M.A. Vijayalakshmi, J. Chromatogr. B Analyt.
Technol. Biomed. Life Sci. 861 (2008) 203.
[21] W.S. Gordon, C.S. David, Rapid Commun. Mass Spectrom. 17 (2003) 1044.
[22] T.B. Stachowiak, T. Rohr, E.F. Hilder, D.S. Peterson, M. Yi, F. Svec, J.M.J.
Frechet, Electrophoresis 24 (2003) 3689.
[23] T. Rohr, E.F. Hilder, J.J. Donovan, F. Svec, J.M.J. Frechet, Macromolecules 36
(2003) 1677.
[24] F. Svec, Electrophoresis 27 (2006) 947.
[25] L. Geiser, S. Eeltink, F. Svec, J.M.J. Frechet, J. Chromatogr. A 1188 (2008) 88.
[26] J. Krenkova, N.A. Lacher, F. Svec, J. Chromatogr. A 1216 (2009) 3252.
[27] T.B. Stachowiak, F. Svec, J.M.J. Frechet, Chem. Mat. 18 (2006) 5950.
[28] J. Krenkova, Z. Bilkova, F. Foret, J. Sep. Sci. 28 (2005) 1675.
[29] Z. Altun, A. Hjelmstroem, M. Abdel-Rehim, L.G. Blomberg, J. Sep. Sci. 30
(2007) 1964.
[30] Z. Altun, L.G. Blomberg, M. Abdel-Rehim, J. Liq. Chromatogr. & Rel.
Technol. 29 (2006) 1477.
[31] T.B. Stachowiak, D.A. Mair, T.G. Holden, L.J. Lee, F. Svec, J.M. Frechet, J.
Sep. Sci. 30 (2007) 1088.
[32] J. Krenkova, N.A. Lacher, F. Svec, Anal. Chem. 81 (2009) 2004.
[33] C. Temporini, E. Perani, F. Mancini, M. Bartolini, E. Calleri, D. Lubda, G.
Felix, V. Andrisano, G. Massolini, J. Chromatogr. A 1120 (2006) 121.
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C h a p t e r 5
Characterization and Application of a Trypsin-
Immobilized Monolithic Polymer with Pipette-
Tip Format for Bioanalysis using LC-MS/MS
5.1 Introduction
In recent years, there has been a significant change in direction in the
pharmaceutical industry and many new drugs are based on biological molecules,
such as proteins and peptides. In testing the efficacy and safety of these new types
of drugs, it is necessary to measure the medication levels in blood or plasma and
the first step in this process is an enzymatic digestion step. Traditional enzyme
digestion protocols present a number of drawbacks, such as long incubation time
and also enzyme autodigestion which results in undesirable formation of
additional peptides, leading to possible ionization suppression in the MS analysis
and interference in the interpretation of the data [1]. Recent major research efforts
have focused on the use of reactive monoliths directly polymerized in different
formats (e.g. capillaries, microchips, columns, pipette tips, syringe needle and
disks), acting as supports for trypsin immobilization. Such immobilized enzyme
techniques have been used in the digestion of proteins, which are followed by MS
determination of the peptides [2-6] and were introduced in Chapter 4 of this thesis.
These applications clearly highlight the potential of enzyme immobilization
technology for fast and efficient sample preparation. However, previous work was
all based on qualitative studies of proteins in standard buffer as a proof of concept
of these enzyme reactors. A limited number of proteomic studies have been
Chapter 5
122
reported for rapid proteolytic digestion and protein identification in serum
samples [3,7]. Conversely, quantitative analyses of proteins are more applicable
and important to the bioanalytical setting of the pharmaceutical industry. One of
the most difficult analytical challenges in the drug discovery process is preparing
samples for reliable quantification of proteins in a fast and efficient manner. These
proteins are potential biomarkers of disease in biological matrices.
Tip-based methods reap numerous advantages in terms of speed, efficiency,
sample clean-up and enrichment with subsequent coupling to LC-MS/MS for high-
throughput bioanalysis. Altun et al. [8] and Abdel-Rehim et al. [9] introduced a
sample preparation technique using a set of polypropylene tips containing a plug
of an in situ polymerized methacrylate-based monolithic sorbent for use with a 96-
tip robotic device. These sorbents have been used successfully for the extraction
and quantification of -blockers and anaesthetics from human plasma by LC–
MS/MS. A novel trypsin-immobilized organic polymer monolith based on a pipette
tip format has been developed and described in Chapter 4 for the digestion of
proteins. The developed IMEPP tips exhibited high hydrolytic activity for the
digestion of cytochrome c, which resulted in very high sequence coverage of over
90% with a short contact time prior to MS analysis.
The aim of this chapter is to explore the utility of tip-based protein digestion
methodologies in a bioanalytical setting. Additionally, this chapter will compare
and contrast the potential advantages of this technology with traditional liquid
phase digestion and commercially available tryptic digest tips, such as MonoTip®
Trypsin. The developed IMEPP tips were characterized using a MicrOTOF-Q
quadrupole time-of-flight MS and LC-MS/MS system including an AB SCIEX
Triple Quadrupole™ 5500 mass spectrometer, followed by an evaluation of its
efficiency in rapid digestion of proteins by running a number of pipetting cycles. In
Chapter 5
123
addition, digestion of proteins spiked in rat plasma was performed for the first
time using enzyme immobilization technology for both qualitative and
quantitative analysis. This method has great potential for high-throughput sample
preparation, enabling a fast quantitative profiling of biological molecules for drug
discovery and development in pharmaceutical industries.
5.2 Experimental
The general experimental details are described in Chapter 2. Detailed conditions
are elaborated in each of the figure captions.
5.2.1 Sample preparation for qualitative analysis
5.2.1.1 Protein digestion in standard buffer
The sample preparation for protein digestion was performed according to the
procedure of Krenkova et al. [10]. Briefly, protein [BSA (bovine serum albumin) or
hIgG (polyclonal human immunoglobulin G)] was dissolved in 100 mM
ammonium bicarbonate, pH 8.2, containing 8 M urea to obtain a solution with a
concentration of 5 mg/mL. The protein was then reduced and alkylated. 1 mL of 5
mg/mL protein was added to 100 µL of 45 mM dl-dithiothreitol (DTT) at 60 C for
40 min. After the temperature of the sample solution was reduced to room
temperature, the proteins were alkylated with 100 µL of 100 mM iodoacetamide
(IAA) for 30 min at room temperature. Before digestion, the reduced and alkylated
protein solution was diluted to 0.5 mg/mL using 50 mM ammonium acetate, pH
8.75 containing 20% acetonitrile. Melittin (0.005 mg/mL, rat calcitonin gene-related
peptide (CGRP) (0.005 mg/mL), cytochrome c (0.1 mg/mL) and myoglobin (0.1
mg/mL) without any previous treatment were dissolved in 50 mM ammonium
acetate, pH 8.75 containing 20% acetonitrile. For protein digestion with soluble
enzyme, denatured protein was diluted with water to achieve a final urea
Chapter 5
124
concentration of 2 M and protein concentration of 1.25 mg/mL (for BSA). Trypsin
was added to 200 µL of protein solution at a substrate-to-enzyme ratio of 50:1
(w/w) for protein digestion and the solution was incubated at 37 °C for 24 h. The
proteolysis was terminated by a pH decrease after the addition of formic acid to a
final concentration of 0.1% (1 µL of 10% formic acid in water). For the digestion of
proteins using IMEPP tips, the protein was dissolved in a solution consisting of 50
mM ammonium acetate, pH 8.75, containing 20% acetonitrile, and 20 pipetting
cycles were used. The peptide fragments from the digestion were collected in
microvials and analyzed using the direct infusion and LC-MS methods. The mass
spectra of all samples were then compared against the protein digestion database
to determine the sequence coverage.
5.2.1.2 Protein digestion in rat plasma
Rat plasma samples were spiked with target proteins (melittin, rat CGRP,
cytochrome c, myoglobin, BSA and IgG) individually to achieve the same
concentration as described above. Then, 1 µL of plasma spiked with target protein
was made up to a total volume of 100 µL using 50 mM ammonium acetate buffer
with 20% acetonitrile for IMEPP tip digestion. The peptides from the digestion
were collected in microvials and analyzed using the direct infusion and LC-MS
methods. The peptide fragments were identified using a database search of Protein
Prospector tool.
5.2.2 Protein identification and multiple reaction monitoring (MRM)
design
5.2.2.1 Sample preparation for MRM development
Different proteins, including melittin, rat CGRP, cytochrome c, myoglobin,
transferrin, BSA and hIgG per 20-µL aliquot of 10 mg/mL were prepared using
digestion buffer (100 mM Tris-HCl/ 10 mM CaCl2/ 20% acetonitrile). Each of the
Chapter 5
125
proteins was digested individually for the development of MRM methods. For
smaller-sized proteins such as melittin, rat CGRP, cytochrome c and myoglobin,
each of the 20 µL aliquot protein samples was diluted with 450 µL of digestion
buffer, 10 µg of trypsin added and the sample were incubated at 37 C for 24 h on
an Eppendorf heating block. For larger-sized proteins that needed to be chemically
treated, such as transferrin, BSA and hIgG, the 20 µL aliquot protein samples were
diluted with 25 µL of 6 M guanidine hydrochloride (GdnHCl). Samples were
reduced by adding 15 µL of 7 mM TCEP, followed by 45-min incubation at 56 °C
on an Eppendorf heating block. Alkylation of cysteine residues was carried out by
adding 15 µL of 13 mM iodoacetamide and incubating the sample for 1 h at 37 C.
Alkylated protein samples were diluted with 450 µL of digestion buffer, 10 µg of
trypsin added and the solutions were incubated at 37 C overnight. Each of the
digested proteins was further diluted 1:80 with the digestion buffer. 75 µL of
protein samples (100 µL for myoglobin) were injected and analyzed by the LC-
MS/MS system.
5.2.2.2 Peptide MRM development
A scheme describing the work flow for the method development of test proteins
and peptides in plasma is shown in Figure 5-1. Peptide fragment masses were
selected from in silico digest of each target protein using the ExPASy tool available
from the website http://au.expasy.org/proteomics. The “Tools” option of the
website was chosen, followed by the selection of “PeptideMass” function. For
smaller-sized proteins (e.g. melittin, rat CGRP, cytochrome c and myoglobin) that
were not reduced and alkylated, the amino acid sequence of each protein was
inserted in the field. Trypsin was chosen as the enzyme for the database search,
with no missed cleavages being allowed. For larger-sized proteins that were
chemically reduced and alkylated, the option “cysteines treated with
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126
iodoacetamide” was chosen. The possible surrogate peptides resulted from the
digestion of each protein were displayed. Due to the possibility of chosen surrogate
peptide in the target standard protein being present in other target proteins or
sample matrix (in this case, rat plasma), each surrogate peptide was checked via
BLASTP searches to ensure that each selected proteotypic peptide corresponded to
a single gene product only.
Due to the limitation in mass range of the API5500 (up to 1000 mAU for the
linear ion trap and 1250 for MRM quantitative mode), the multiply charged parent
ions (mainly +2 & +3) were utilized for peptides larger than 1000 Da. An enhanced
product ion (EPI) scan was then utilized, for up to five parent ions simultaneously.
The EPI scan enabled the identification of the product ions with highest intensity
and suitable collision energy to construct a quantitative (MRM) method.
Figure 5-1: Schematic for the method development of test proteins and peptides
in rat plasma.
5.2.3 Characterization of performance of IMEPP tips
5.2.3.1 Plasma loading experiment for IMEPP tips
Chapter 5
127
The rat plasma sample was spiked with a mixture of four target proteins
(i.e. melittin, rat CGRP, cytochrome c and myoglobin) to achieve a final
concentration of 100 µg/mL for each protein. Different amount of rat plasma
ranging from 1 – 60 µL (1, 2.5, 5, 10, 20, 40, 60 µL) were made up to a total volume
of 170 µL with the digestion buffer and digested using IMEPP tips. The digestion
protocols used for commercially available tryptic digest MonoTip® Trypsin and
IMEPP tips were described previously in Section 2.3.9. For liquid phase digestion,
trypsin (10 µg) was added to each sample solution and the solution was incubated
at 37 C for 24 h. The proteolysis was terminated by a pH decrease after the
addition of formic acid to a final concentration of 0.1% (1 µL of 10% formic acid in
water). The resultant peptide solutions were made up to a total volume of 200 µL
using mobile phase A (5% acetonitrile/ 95% water with 0.1% formic acid) and were
injected onto an AB SCIEX Triple Quadrupole™ 5500 mass spectrometer using the
multiple reaction monitoring (MRM) mode. Extracted ion chromatograms (EIC)
corresponding to tryptic digested peptides of target proteins were recorded and
areas of the eluting peaks were computed.
5.2.3.2 Time course assay
Rat plasma spiked with four target proteins (100 µg/mL for each protein) was used
in this assay. A 10 µL sample of the plasma was diluted with 160 µL of digestion
buffer. The protein sample solution was digested using IMEPP tips and MonoTip®
Trypsin for a duration of 30 min. Different digestion durations of 30 min, 1 h, 4 h
and 24 h were set up for liquid phase digestion of the protein sample, while
keeping the amount of trypsin (10 µg) constant. The digested samples were diluted
1:20 using the digestion buffer and analyzed with the LC-MS/MS system.
5.2.3.3 Sample clean-up and enrichment functionality assay
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128
The rat plasma spiked with four target proteins (100 µg/mL for each protein) was
again used in this assay. A 10 µL sample of the plasma was diluted with 160 µL of
digestion buffer, one with and one without 20% acetonitrile. Then, the plasma
samples were digested with IMEPP tip and MonoTip® Trypsin. The digestion tips
using 100% aqueous buffer were eluted with 50 µL of 100% acetonitrile containing
0.1% formic acid. Sample solutions digested in digestion buffer with and without
20% acetonitrile as well as the eluted samples from the aqueous-digestion-tips
were analyzed using the LC-MS/MS system.
5.2.3.4 Volume test
The rat plasma (10 µL) spiked with four target proteins (100 µg/mL for each
protein) was diluted with 60 µL and 160 µL of digestion buffer. Then, plasma
samples with different total volumes (70 µL and 170 µL) were digested with
IMEPP tips and analyzed using the LC-MS/MS system.
5.2.4 Sample preparation for quantitative analysis in rat plasma
For quantitative analysis, the rat plasma sample was spiked with a mixture of
four target proteins (i.e. melittin, rat CGRP, cytochrome c and myoglobin) to
achieve final concentrations of 1.5625, 3.125, 6.25, 12.5, 25 and 50 µg/mL for each
protein in plasma. To each protein mixture with different concentration, 10 µL of
plasma was added to 60 µL of buffer (100 mM Tris-HCl/ 10 mM CaCl2/ 20%
acetonitrile) and the sample digested using IMEPP tips. The digested sample
solutions were back-diluted to a volume of 200 µL using mobile phase A (5%
acetonitrile/ 95% water with 0.1% formic acid).
For larger-sized proteins that need to be chemically treated, such as
transferrin, BSA and hIgG, the rat plasma samples were spiked with a mixture of
three target proteins to achieve final concentrations of 1.5625, 3.125, 6.25, 12.5, 25
and 50 µg/mL for each protein in plasma. The high-molecular weight proteins
Chapter 5
129
spiked in plasma (10 µL) were diluted with 15 µL of Tris-HCl, pH 8.8 containing
10 mM of CaCl2 and 20% acetonitrile, and were further diluted with 25 µL of 6 M
(GdnHCl). Samples were reduced and alkylated. Alkylated protein samples were
desalinated with 420 µL of the same buffer. Aliquots of 100 µL of the reduced
and alkylated protein solutions were pipetted with 20 cycles of pipetting
procedure and the tip was soaked in the sample solution for 30 min. The
resultant peptide solutions were diluted with 100 µL buffer and analyzed by the
AB SCIEX Triple Quadrupole™ 5500 mass spectrometer using the MRM mode.
A calibration curve was constructed based on the peak areas recorded against the
concentration of proteins spiked in rat plasma.
5.3 Results and discussion
5.3.1 Application of IMEPP tips to the digestion of model proteins
spiked in standard buffer and rat plasma
The high complexity of plasma makes the digestion of proteins using the
immobilized enzyme method very challenging. Tens of thousands of proteins are
present in plasma and are dispersed over an extremely wide concentration range.
Several different proteins have often been used in model test systems that include a
proteolytic digestion step. In this study, six proteins with molecular masses
ranging from 2.8 kDa to 150 kDa were used as the model proteins. Under
optimized conditions, the excellent proteolytic activity of the IMEPP tips on these
proteins was demonstrated in Chapter 4. Due to the low target substrate levels that
are present in plasma, trypsin autolysis becomes a significant competing reaction
which reduces substrate conversion, and the reaction is substrate-limited. In
addition, the non-specific adsorption of proteins may also block the active sites on
the IMEPP tips and decrease the tryptic digest activity. The newly developed
Chapter 5
130
method, involving the immobilization of enzyme on a large through-pore organic
polymer monolith support using higher enzyme concentration, enhanced the
hydrolysis rate and enabling rapid diffusion, should promote efficient digestion of
low concentration substrates. This is the first time a rapid digestion of proteins
spiked in plasma using the IMEPP tips developed has been performed.
The high efficiency digestion of the small protein rat CGRP using the IMEPP
tip is shown in Figure 5-2. The large protein peak was missing after the digestion.
The IMEPP tip also showed very high proteolytic activity even when it was used to
digest the protein spiked in rat plasma sample (Figure 5-2). No significant
difference was found between the digestion using soluble trypsin and IMEPP tips
(Figure 5-2). Figure 5-3 shows base peak chromatograms (BPC) of cytochrome c
digest prepared using 24 h digestion with soluble trypsin, with 15 pipetting cycles
(~ 6 min) for digestion of cytochrome c in digestion buffer and rat plasma using
IMEPP tips at a concentration of 0.1 mg/mL (Figure 5-3). No marked difference
between the digestions using soluble trypsin, IMEPP tip and digestion in rat
plasma using IMEPP tip was observed (Figure 5-3). However, some cytochrome c
remained undigested when in-solution digestion was used (Figure 5-3). By
contrast, the IMEPP tip exhibited excellent performance, with complete digestion
of cytochrome c and much faster digestion in comparison with the conventional in-
solution digestion (Figure 5-3). It is also worth mentioning that the liquid phase
digestion of protein spiked in rat plasma would result in undesirable formation of
additional peptides due to enzyme autodigestion, which may lead to ionization
suppression or interference in the interpretation of data (Figures 5-2 and 5-3).
Meanwhile, it has been demonstrated that using tip-based protein digestion, the
problem of enzyme autodigestion was eliminated (Figures 5-2 and 5-3).
Chapter 5
131
Figure 5-2: Chromatograms of a tryptic digest of rat CGRP (0.005 mg/mL) using the IMEPP tip and soluble trypsin in
both standard buffer and rat plasma. Conditions: HPLC column, Dionex Acclaim® PolarAdvantge C16 (2.1 x 100 mm, 3
µm); temperature, 30 °C; flow-rate, 0.2 mL/min; injection volume, 5 µL; mobile phases: A, 0.1% formic acid in water; B,
0.1% formic acid in acetonitrile; a linear gradient of B% from 20% to 55% in 35 min.
Chapter 5
132
Figure 5-3: Chromatograms of a tryptic digest of cytochrome c (0.1 mg/mL) using the IMEPP tip and soluble trypsin in
both standard buffer and rat plasma. Conditions are the same as described in Figure 5-2.
Chapter 5
133
Cytochrome c and other protein standards were chosen because they are
inexpensive, stable and easy to prepare due to the lack of cysteines which
simplifies the digestion protocols. Therefore, any specific treatment prior to
digestion was not necessary when small proteins such as rat CGRP or cytochrome
c were used. On the other hand, the three-dimensional protein structure of large
proteins, such as BSA, was stabilized by chemical interactions such as disulfide
bonding, and RP or hydrophilic interactions between the polypeptide chains. In the
case of digesting BSA with the IMEPP tip, pre-treatments such as chemical and
thermal were required. Without any specific pre-treatment prior to digestion, the
IMEPP tip would be unable to catalyze digestion, since the active site of the trypsin
did not come into contact with the peptide chain within shorter digestion times [4].
Therefore, the process of chemical reduction and alkylation are generally
employed to enhance exposure sites of the protein which were previously
inaccessible for proteolysis [11]. In this study, the salts present in the protein
solution after this treatment were diluted 10 times so that the proteolytic activity of
the immobilized enzyme would not be compromised. Figure 5-4 shows the LC-MS
separation of the digestion of denatured BSA with the IMEPP tip. The digested
peptide was obtained with 15 pipetting cycles at 37 C. The peptide profile
digested with the developed tip was the same as that processed with the
conventional method.
The individual digests of the proteins in standard buffer and those that were
spiked in rat plasma are shown in Figures 5-5 and 5-6. The digestion was both very
fast and efficient and no real difference was observed between mass spectra
obtained from the in-solution digestion (not shown) and the digestion using
IMEPP tips for the digestion of rat CGRP, cytochrome c and BSA. In addition, the
IMEPP tips were also used for the digestion of standard proteins, melittin and
Chapter 5
134
Figure 5-4: Chromatograms of a tryptic digest of BSA (0.5 mg/mL) using the IMEPP tip and soluble trypsin in both
standard buffer and rat plasma. Conditions are the same as described in Figure 5-2 except a linear gradient of B% from
10% to 50% B in 35 min.
Chapter 5
135
+MS, 1.0-1.9min #(57-114)
0
20
40
60
80
100
Intens.
[%]
250 500 750 1000 1250 1500 1750 2000 2250 m/z
+MS, 0.3-1.4min #(16-84)
0
20
40
60
80
100
Intens.
[%]
250 500 750 1000 1250 1500 1750 2000 2250 m/z
Melittin(0.005 mg/mL)2.8kDa
Calcitonin Gene Related Peptide rat (0.005 mg/mL)3.8kDa
+MS, 0.5-1.0min #(41-78)
0
20
40
60
80
100
Intens.
[%]
250 500 750 1000 1250 1500 1750 2000 2250 m/z
Cytochrome c (0.01 mg/mL)13kDa
+MS, 0.3-0.4min #(15-23)
0
20
40
60
80
100
Intens.
[%]
250 500 750 1000 1250 1500 1750 2000 2250 m/z
BSA(0.5 mg/mL)66kDa
+MS, 0.2-0.3min #(9-19)
0
10
20
30
Intens.
[%]
250 500 750 1000 1250 1500 1750 2000 2250 m/z
hIgG(0.5 mg/mL)150kDa
+MS, 0.3-0.7min #(18-39)
0
20
40
60
80
100
Intens.
[%]
250 500 750 1000 1250 1500 1750 2000 2250 m/z
Myoglobin(0.1 mg/mL)17kDa
Figure 5-5: ESI-TOF MS spectra of peptides obtained by digestion of six proteins using IMEPP tips. Conditions: direct
infusion flow-rate, 180 µL/hr; acquisition time, 2 min; capillary voltage, 4.5 kV; end plate offset, 500 V; nebulizer, 0.3
bar; drying gas, 4 L/min; temperature, 180 °C. Asterisks define peaks of positively identified peptides after protein
digestion.
Chapter 5
136
+MS, 0.3-0.4min #(15-24)
0
20
40
60
Intens.
[%]
250 500 750 1000 1250 1500 1750 2000 2250 m/z
Calcitonin Gene Related Peptide rat (0.005 mg/mL)
+MS, 0.3-0.5min #(18-30)
0
20
40
60
80
Intens.
[%]
250 500 750 1000 1250 1500 1750 2000 2250 m/z
Cytochrome c (0.01 mg/mL)
+MS, 0.4-0.6min #(25-37)
0
20
40
60
80
100
Intens.
[%]
250 500 750 1000 1250 1500 1750 2000 2250 m/z
+MS, 0.2-0.3min #(14-18)
0
5
10
15
Intens.
[%]
250 500 750 1000 1250 1500 1750 2000 2250 m/z
hIgG (0.5 mg/mL)
BSA(0.5 mg/mL)
Figure 5-6: ESI-TOF MS spectra of peptides obtained by digestion of four proteins spiked in rat plasma using IMEPP
tips. Conditions are the same as described in Figure 5-5.
Chapter 5
137
myoglobin, by direct infusion ESI-TOF MS method (Figure 5-5). The mass spectra
of all samples were then compared against the protein digestion database (protein
prospector, http:// prospector.ucsf.edu/) to identify the digest and determine the
sequence coverage. Asterisks were not included in the digestion of hIgG in Figure
5-6 because this protein contains six different components. It would be difficult to
differentiate the peptides resulting from the digestion of these components using
asterisks. Table 5.1 summarizes the results of highest sequence coverage obtained
for each digested protein. An ideal coverage of 100% was achieved for the low-
molecular mass peptide rat CGRP using soluble enzyme and the IMEPP tip. In
addition, the sequence coverages of the proteins digested using IMEPP tips were
above 88% in standard buffer and also above 84% for proteins spiked in rat plasma
(Table 5.1).
A myoglobin trypsin digest always results in relatively few peptides and
exhibits large protein peaks at high concentration (0.5 mg/mL), as shown in Figure
5-7 (a). This is because myoglobin contains a heme group which is hard to digest.
The early studies of Russell et al. showed that enzymatic digestion in mixed
organic-aqueous solvent systems were highly efficient in terms of both the rate of
digestion and amino acid sequence coverage [12]. They reported that trypsin
maintained proteolytic activity in a variety of solvents (e.g. methanol, acetone, 2-
propanol and acetonitrile) using up to 80% organic solvent [12]. Several authors
have also confirmed that the activity of immobilized trypsin is not affected by
organic solvents, with the usage of up to 100% acetonitrile [13,14]. Gordon and
David also reported an optimum of 45% acetonitrile used to achieve the highest
sequence coverage of the digestion [14]. Therefore, a digestion buffer containing
45% or 80% acetonitrile in 50 mM ammonium acetate, pH 8.75 was used for
myoglobin digestion in order to improve the digestion efficiency. Surprisingly, the
Chapter 5
138
Table 5.1: Results of sequence coverage for the digestion of proteins and spiked samples using IMEPP tips.
Protein MW (Da)Concentration
(mg/mL)
Sequence Coverage (%)Soluble trypsin
IMEPPIMEPP (Spiked
sample)Melittin 2848 0.005 100 100 100
Rat CGRP 3806 0.005 100 100 100Cytochrome c 11702 0.01 97 95 87
myoglobin 16951 0.1 95 89 66BSA 69294 0.5 92 88 84
Chapter 5
139
186.9
276.5
340.0
397.0
454.0
543.4
631.0
680.1
749.0
790.0
851.5
892.7
941.0 997.6
1060.01130.6
1211.3
1304.4
1413.01541.5 1695.5
1883.8
1952.3
2119.3
2196.2
2355.8
+MS, 1.3-2.7min #(95-199)
0
10
20
30
40
50
Intens.
[%]
250 500 750 1000 1250 1500 1750 2000 2250 2500 m/z (a)
186.9276.5
340.0
454.0
615.8 680.1
753.6
807.8
848.1
892.7
942.3
997.6
1060.0
1130.6
1211.3
1304.4
1413.0
1541.4 1695.5
1883.8
1952.2
2020.7
2119.3
2196.2
2355.8 2421.8
2509.7
+MS, 1.0-2.2min #(74-166)
0
10
20
30
40
50
Intens.
[%]
250 500 750 1000 1250 1500 1750 2000 2250 2500 m/z (b)
Figure 5-7: ESI-TOF MS spectra of peptides obtained by digestion of myoglobin
(0.5 mg/mL) using IMEPP tips in (a) 20% acetonitrile (sequence coverage: 97%),
(b) 45% acetonitrile (sequence coverage: 88%) in digestion buffer.
Chapter 5
140
sequence coverage obtained from the digestion of myoglobin using 45%
acetonitrile was lower than using 20% acetonitrile [Figure 5-7 (b)].
Several reports have suggested that using high organic solvents might
increase the proteolytic activity of the trypsin, since increasing the amount of
organic solvent also increases the solubility of the substrate, which changes the
conformation of substrate or enzyme itself, providing more accessible active sites
for the substrate [12]. One thing that is known with certainty is that increasing the
amount of acetonitrile also increases the eluting strength of the solution, enabling it
to release more digested peptides from the monolith support and thus allowing
more digested fragments to be detected. A similar value of sequence coverage was
obtained despite using 45% acetonitrile in the digestion buffer for BSA digestion.
Using 80% acetornitrile in the digestion buffer was also tried for the digestion of
BSA. However, a higher percentage of acetonitrile resulted in the precipitation of
the protein which blocked the IMEPP tip. Furthermore, it is important to note that
the lowest concentration of acetonitrile in the digestion buffer is always preferable
to facilitate the subsequent gradient separation of peptides in reversed-phase
chromatography (e.g. LC-MS). Therefore, 20% acetonitrile was employed for the
digestion of high-molecular weight protein.
The efficiency of the trypsin-immobilized organic polymer pipette tip was
also evaluated with high-molecular weight proteins, such as human polyclonal
IgG. The rapid growth of antibody drugs and drug candidates in the
biopharmaceutical industry has created a demand for automated proteolytic
digestion to assist in pharmaceutical stability studies, to identity assays and to
facilitate quality control of these therapeutic proteins [15]. Therefore, efficient and
rapid digestion is necessary for studying this extensive and complex protein. This
protein contains four types of heavy chains (Igg-1, Igg-2, Igg-3 and Igg-4) and two
Chapter 5
141
Figure 5-8: Chromatograms of a tryptic digest of hIgG (0.5 mg/mL) using the IMEPP tip and soluble trypsin in both
standard buffer and rat plasma. Conditions are the same as described in Figure 5-4.
Chapter 5
142
types of light chains (Igg-kappa and Igg-lambda). Again, this high-molecular
weight protein has to be reduced and alkylated prior to the in-solution and IMEPP
tip digestion. The peptide chromatogram of the digestions is shown in Figure 5-8.
The mass spectra of the digests of hIgG using soluble trypsin and the IMEPP tip in
both standard buffer and rat plasma are shown in Figures 5-5 and 5-6. Results
characterizing the digestion of hIgG chains obtained using both soluble trypsin and
the IMEPP tip are summarized in Table 5.2. Similar values of sequence coverage
were observed for both the trypsin-immobilized pipette tip and use of the soluble
enzyme. However, lower sequence coverage was observed for the digestion of
protein spiked in rat plasma. This might be due to the high protein components
which were present in rat plasma, blocking the majority of active sites on the
trypsin-immobilized monolith support, thereby resulting in a loss of activity of the
IMEPP tip during the digestion of heavy chains (i.e. Igg-1) [16]. However, this
method works well for the light chains (i.e. Igg-kappa and Igg-lambda), yielding
sequence coverage similar to that of the in-solution digestion, albeit with an
additional advantage of very short digestion time (~ 6 min). In summary, these
results suggest that the developed trypsin-immobilized monolithic polymer tips
can be used for fast and efficient digestion of both low- and high-molecular weight
proteins, thus indicating that this tip can be used for proteome studies.
5.3.2 Design of MRM assays for target proteins
The application of MRM to peptide analysis has created a new breakthrough for
protein analysis in complex biological matrices. Due to the multiple fragment steps
in MRM, higher selectivity is achieved. In this chapter, MRM methods were
developed in order to identify the peptides after protein digestion, which can be
used to interpret the digestion efficiency. Specifically, a range of different assays
were developed including plasma loading capacity, time course assay, sample
Chapter 5
143
Table 5.2: Comparison of sequence coverage identification of hIgG using soluble trypsin, IMEPP tips and IMEPP tips for
spiked samples.
Chapter 5
144
clean-up and effect of digestion volume for the evaluation of the digestion
efficiency of the developed IMEPP tips in comparison with commercially available
tryptic digest tips and liquid phase digestion.
To develop a targeted analysis method, proteotypic peptides from each
protein were selected for detailed analysis and MRM transition design. The
molecular weight of cytochrome c is 11701.55 Da; its singly charged molecular ion
could not be observed by the LC-MS/MS because it is above the mass detection
range of the instrument. Therefore, surrogate peptides obtained from the tryptic
digest of this protein had to be used to represent the amount of the protein present
in the sample matrix. An example of in silico digest of cytochrome c from the
equine heart is given in Figure 5-9. The surrogate peptides at different charged
states were calculated using the in-house Peptide Mass Calculator. The entire
surrogate peptides were BLAST searched against the sample matrix (in this case,
rat plasma) to look for uniqueness in these surrogate peptides. For each protein,
two or more proteotypic peptides were selected for further analysis. Care was
taken where possible to ensure the selected peptides did not contain cysteine or
methionine residues and were between 8 and 15 amino acids in length [17]. From
Figure 5-10, it can be observed that the surrogate peptide with amino acid
sequence EETLMEYLENPK only existed in equine heart cytochrome c and not in
any rat genome sequences. This therefore can potentially be used as a surrogate
peptide for quantification of equine heart cytochrome c in rat plasma.
The unique surrogate peptides of cytochrome c were selected as the first
precursor ions in the product ion scan mode. The precursor ions were set into the
EPI mode (normally up to five ions) in order to identify the most intense fragment
ions with suitable collision energy to construct a quantitative (MRM) method
(Figure 5-11). For each peptide, two or more transitions were selected from the
Chapter 5
145
Figure 5-9: A typical result of in silico digest of cytochrome c.
Figure 5-10: BLAST search result of horse heart cytochrome c against rat genome.
Chapter 5
146
Figure 5-11: EPI experiment spectra from cytochrome c.
Chapter 5
147
remaining transitions based on the most intense y ions in the MS/MS spectra, with
preference given to product ions with m/z values above the precursor ion to
increase MRM specificity. In this case, the major fragment ions produced from the
doubly-charged ion (m/z = 748.35) are m/z = 892.5, 763.4 and 600.3 (Figure 5-12).
The fragment ions were checked using “MS-product” in the protein prospector to
ensure that they were from the precursor ion. Table 5.3 shows that the fragment
ions m/z = 892.5, 763.4 and 600.3 corresponded to y7, y6, and y5 ions of the
surrogate peptide. To decrease the MS duty cycle while ensuring a high number of
MS/MS scans over peak, only the most intense MRM transition for each peptide
was kept in the final method, and the MS/MS conditions for these MRMs were
optimized. Therefore, the most intense fragment m/z 892.5 was selected as the
transition for one of the surrogate peptides for the quantification of cytochrome c
for different assays and quantitative analysis (Figure 5-12). The same procedure
was carried out for all the standard proteins (melittin, rat CGRP, myoglobin, BSA,
transferrin, hIgG). Details of the conditions used for the analysis of each peptide,
including optimized transition parameters and collision energies for targeted
proteins are shown in Tables 5.4 and 5.5. In total, the list indexes eight proteins,
with a total number of 22 surrogate peptides. Figure 5-13 shows a typical LC-
MS/MS chromatogram with MRM transitions of protein mixture (melittin, rat
CGRP, cytochrome c and myoglobin).
5.3.3 Characterization of the digestion performances of IMEPP tips in
Pfizer UK
To evaluate the digestion performances of IMEPP tips, the following assays were
considered in particular: the plasma loading capacity, digestion efficiency over
different durations, sample enrichment functionality and the optimum sample
Chapter 5
148
TIC of +EPI (748.35) CE (35): Exp 1, from Sample 1 (Cyto 5EPIa bulk digest 200ug diluted 1 to 1 inj 25uL) of 12_07_2010_EPI_CYTO.wiff (Turbo ... Max. 2.7e8 cps.
2 4 6 8 10 12 14 16 18 20 22 24 26Time, min
0.0
2.0e7
4.0e7
6.0e7
8.0e7
1.0e8
1.2e8
1.4e8
1.6e8
1.8e8
2.0e8
2.2e8
2.4e8
2.6e8
2.7e8
Inte
ns
ity
, c
ps
11.01
+EPI (748.35) CE (35): Exp 1, 10.826 to 11.182 min from Sample 1 (Cyto 5EPIa bulk digest 200ug diluted 1 to 1 inj 25uL) of 12_07_2010_EPI_CY... Max. 1.7e6 cps.
200 250 300 350 400 450 500 550 600 650 700 750 800 850 900 950 1000m/z, Da
0.0
1.0e5
2.0e5
3.0e5
4.0e5
5.0e5
6.0e5
7.0e5
8.0e5
9.0e5
1.0e6
1.1e6
1.2e6
1.3e6
1.4e6
1.5e6
1.6e6
1.7e6
Inte
ns
ity
, c
ps
892.5
763.4
455.2342.1
600.3
358.2
244.2
231.1
586.2427.2409.2 487.3437.2
715.3 748.3324.2296.1 512.2896.3226.1 473.3 874.3259.1 576.3
604.3 730.3568.8280.2 619.3445.1 666.2 991.3503.3213.1 697.3245.1 388.0306.1 374.2 455.8 767.3 860.3722.0541.3 803.2 848.3301.1 582.4 987.4683.8652.3591.2428.3 909.3915.7
Figure 5-12: MRM methods workflow by selecting the most intense fragment
ions from the product ion of cytochrome c. (A) shows the single product ion m/z
748.35 from the EPI experiment spectra. (B) shows the MS/MS fragment ions of
the selected product ion.
A
B
JasonHon
Rectangle
JasonHon
Rectangle
Chapter 5
149
Table 5.3: Theoretical peak table generated from the surrogate peptide m/z 748.35 of cytochrome c.
Chapter 5
150
Table 5.4: Details of surrogate peptides, Q1, Q3 mass and specific collision energy for each target protein.
Protein MRM transitions
Protein Peptide Sequence Peptide mass Charge state Q1 Q3 Collision Energy
Melittin VLTTGLPALISWIK 1511.9 2+ 756.8 927.7 30
Rat CGRP DNFVPTNVGSEAF 1396.6 2+ 698.8 476.3 20
Myoglobin GLSDGEWQQVLNVWGK 1815.9 2+ 908.4 390.3 40
VEADIAGHGQEVLIR 1606.9 2+ 803.9 814.5 45
HGTVVLTALGGILK 1378.8 2+ 689.8 885.5 40
LFTGHPETLEK 1271.7 2+ 636.3 716.3 40
HPGDFGADAQGAMTK 1501.7 2+ 751.8 742.8 37
Cytochrome c EETLMEYLENPK 1495.7 2+ 748.3 892.4 30
TGQAPGFTYTDANK 1470.7 2+ 735.8 358.2 35
TGPNLHGLFGR 1168.6 2+ 584.8 549.3 35
EDLIAYLK 963.5 2+ 482.8 494.3 20
Chapter 5
151
Table 5.5: Details of surrogate peptides, Q1, Q3 mass and specific collision energy for each target protein that was
chemically treated.
Protein MRM transitions
Protein Peptide Sequence Peptide mass Charge state Q1 Q3 Collision Energy
BSA QTALVELLK 1014.6 2+ 507.8 785.5 25
LVNELTEFAK 1163.6 2+ 582.3 951.5 25
DAFLGSFLYEYSR 1567.7 3+ 523.3 717.3 25
Transferrin YLGEEYVK 1000.5 2+ 500.8 724.3 25
SVIPSDGPSVACVK 1415.7 2+ 708.4 558.8 35
IgG-Kappa SGTASVVCLLNNFYPR 1797.9 2+ 899.0 435.3 35
TVAAPSVFIFPPSDEQLK 1946.0 3+ 649.3 913.4 25
VYACEVTHQGLSSPVTK 1875.9 3+ 626.0 807.3 25
IgG1 FNWYVDGVEVHNAK 1677.8 3+ 559.9 708.8 25
GPSVFPLAPSSK 1186.6 2+ 593.8 699.5 30
Chapter 5
152
Figure 5-13: LC-MS/MS chromatogram corresponding to the MRM transitions associated with specific peptides for each
targeted protein.
Chapter 5
153
volume for digestion. All the assays were completed using the MRM methods
developed previously.
5.3.3.1 Plasma loading capacity of IMEPP tips
The plasma loading experiment was designed to determine the maximum amount
of plasma that can be used to yield the highest efficiency for digestion. This is
particularly important since, if a lower amount of plasma sample is used, the
sensitivity of detection will decrease; if too much plasma is used, it will block the
tips and also decrease the tryptic digestion efficiency (due to the relative amount of
protein present in the sample). This plasma loading experiment was also used to
compare the sample capacity when liquid phase digestion and tip-based
technology (i.e. MonoTip® Trypsin and the IMEPP tip) were used. Figure 5-14
shows one of the examples of plasma loading capacity for liquid phase digestion
compared to the two different tip-based technologies. Liquid phase digestion
always gives lower plasma capacity, in this case 5-10 µL of plasma, due to the
limited amount of trypsin used for digestion in a bid to minimize auto-digestion
that can potentially interfere with the sensitivity of detection.
Immobilized enzyme technology especially for tip-based methods, offers
the advantages of high plasma loading capacity of up to 40 µL of plasma, fast
digestion rate, and the ability to use a high concentration of enzyme for digestion
without compromising the detection. This is consistent with all the other tested
proteins. The present study also showed that the developed IMEPP tips based on a
porous polymer monolith exhibited higher capacity and efficiency than the
commercially available tryptic digest tips based on a silica monolith acting as
enzyme support. Enzyme immobilization on a porous polymer monolith offers
distinct advantages over the silica monolith in terms of flexibility of synthesis,
greater biocompatibility over a wide pH range and also versatile functionality
Chapter 5
154
Figure 5-14: Plasma loading capacity for liquid phase digestion, MonoTip®
Trypsin and IMEPP tips for the digestion of cytochrome c spiked in different
amount of rat plasma.
Chapter 5
155
modification. The highly porous surface facilitates the flow-through of the sample
solutions and moreover, the stability of the polymer support for trypsin
immobilization means that polymer monolith will not shrink even with up to 40
µL of plasma used for the digestion.
5.3.3.2 Time course study
A time course study was set up in order to investigate the efficiency of digestion
for the IMEPP tip for different durations compared to the commercially available
enzyme-tip and traditional in-solution digest. Figure 5-15 shows clear
predominance of the newly developed IMEPP tips compared to the commercially
available product and liquid phase digest over a 30 min digestion period. When
compared to the in-solution digest analyses, the 30 min IMEPP tip digest yielded
results similar to those of the 24 h solution digest.
Figure 5-15: Digestion time course of cytochrome c with tip-based digestion (30
min) compared to commercially available MonoTip® Trypsin and in-solution
digestion (30 min, 1 h, 4 h and 24 h).
Chapter 5
156
5.3.3.3 Sample clean-up and enrichment functionality assay
Given the very small sizes of the samples handled in proteomics, miniature
immobilized enzyme reactors placed in capillaries or microfluidic devices have
been developed. However, the identification of proteins remains a challenge as the
proteins are present in solutions at very low concentrations. The other significant
challenge is that matrix interferences such as salts, buffers and detergents must be
eliminated prior to the LC-MS/MS analyses. In order to obtain reliable analytical
results using these techniques, sample pre-treatment and pre-concentration using
solid-phase extraction (SPE) can be employed [18]. The compounds of interest are
first absorbed onto the surface of porous materials and later released by elution
with a strong solvent in a concentrated band. Custom-designed SPE devices have
also been used to eliminate interfering compounds. A dual-function
microanalytical device with the integration of SPE and enzymatic digestion for
peptide mapping has been developed by Peterson et al. [13] . The samples were
pre-concentrated in the first segment of the device, then eluted and digested in the
second segment.
The immobilized enzyme polymer monolith contains hydrophobic
functionality which can be used for SPE. Therefore, the effects of sample
enrichment functionality on IMEPP tips were further studied, as shown in Figure
5-16. The recoveries rapidly decreased for the digestion in 100% aqueous buffer. In
addition, the protein solution eluted from the aqueous digestion tip also showed a
significant drop in recovery. Therefore, the sample enrichment functionality of the
IMEPP tips was not very efficient. Digestion using buffer with 20% acetonitrile
performed better than that obtained using an aqueous. The low recovery was
possibly due to the properties of the monolith used. In this case a higher amount of
cross-linking monomer was used to obtain a highly porous material. The
Chapter 5
157
functional monomer was employed at a lower concentration of hydrophobic
monomer, resulting in a loss of the hydrophobic functionality. It is recommended
that a formulation should be used with a higher amount of functional monomer or
cation-exchange monomer for future studies. However, as discussed in Section
5.3.1, the tips still provide great potential for better sample clean-up and higher
sensitivity of detection without the problem of enzyme autodigestion.
Figure 5-16: Peak area of surrogate peptide of cytochrome c digested using
MonoTip® Trypsin and IMEPP tips using buffer/20% acetonitrile, aqueous and
the eluted samples from the aqueous digestion-tips.
Chapter 5
158
5.3.3.4 Volume test
The volume test was employed to compare different dilutions for the same amount
of plasma used for digestion to suit different purposes. For example, a volume just
enough for a tip was used to have a fair comparison so that all the solution has the
same residency digestion time on the stationary phase. If the total digestion
volume used is 170 µL, it is expected to take 3-4 pipetting cycles for all solution to
pass through the enzyme immobilized surface. Alternatively, we can pass all the
solution through the immobilized bed if a smaller digestion volume of 70 µL used.
One concern is that the concentration of plasma in solution might block the tip if a
lower digestion volume used (also because of the lower dilution factor).
Optimization steps will therefore be expected if different digestion volumes need
to be used. In this case, 10 µL of plasma was used and diluted with 170 µL
digestion buffer compared to 70 µL of digestion buffer for digestion in tips. Both of
the sample solutions were then back-diluted to 200 µL using mobile phase A after
digestion using IMEPP tips.
Figure 5-17 shows the recoveries for the surrogate peptides of four different
proteins after digestion using an IMEPP tip for digestion volumes of 170 µL and 70
µL. It appears that the overall recoveries of surrogate peptides are higher using the
70 µL digestion volume. The most significant results can be seen for surrogate
peptides MELITTIN_A, RAT CGRP_B and CYTO_F (Figure 5-17). However, the
recoveries for the peptides from myoglobin were relatively low. This is expected
due to the presence of heme groups which are hard to digest. As discussed earlier,
the higher digestion efficiency when using a lower digestion volume could be due
to increased exposure of target proteins to the enzyme immobilized solid support.
Therefore, a digestion volume for tip digestion of 70 µL was chosen for further
studies.
Chapter 5
159
Figure 5-17: Recoveries of surrogate peptides from four different proteins digested on IMEPP tips using different
volumes for digestion.
Chapter 5
160
5.3.4 Quantitative analysis of target proteins using LC-MS/MRM
method after digestion using IMEPP tips
To demonstrate the capability of the developed IMEPP tips to accurately quantify
proteins and peptides in biological samples, quantitative MRM assays were
specifically designed for tryptic digested peptides using IMEPP tips for seven
targeted proteins spiked in rat plasma. MRM methods have been developed in
order to study four simple proteins (i.e. melittin, rat CGRP, cytochrome c and
myoglobin) and three larger proteins that had to be chemically reduced and
alkylated [i.e. BSA, transferrin and hIgG components (Igg-1 and Igg-kappa)]. The
two different groups of proteins were spiked into rat plasma at varying
concentrations and digested using IMEPP tips. The multiplexing capability of LC-
QqQ-MS platforms for measuring peptides in complex digests is substantial,
proving an opportunity to measure large panels of proteins accurately in each run.
The protein levels were monitored to test the sensitivity and linearity of digested
surrogate peptides using the IMEPP tips, as selected through the LC-MS/MRM
method. Quantitative analysis information on six different proteins and the IgG
components at six different concentration levels are reported in Table 5.6. Results
show that the ratio of protein concentration and peak area correlation was linear at
all concentration values studied, with correlation coefficient (R2) values of at least
0.9884. Based on the results presented here, numerous targeted proteins ranging
from low-molecular weight to those of high-molecular weight can be rapidly and
efficiently digested and also quantitatively analyzed using peptide MRMs. It is also
worth mentioning that the quantitative analysis of proteins digested using
developed IMEPP tips demonstrates relatively good linearity over a wide range
without the need for an additional internal standard to be used.
Table 5.6: Quantitative information of the targeted proteins.
Chapter 5
161
Proteins Plasma
used (µL)
Initial conc.
(µg/mL)
Analysis Conc.
(ng/mL)
Correlation
coefficients (R2)
Melittin 10 0.01-10 0.5 - 500 0.9993
Rat CGRP 10 0.01-10 0.5 - 500 0.9970
Cyto c 5 1.56 – 50 40 - 1250 0.9987
Myoglobin 5 1.56 – 50 40 - 1250 0.9985
BSA 1 1.56 – 50 8 - 250 0.9925
Transferrin 1 1.56 – 50 8 - 250 0.9898
Igg-1 1 1.56 – 50 8 - 250 0.9884
Igg-kappa 1 1.56 – 50 8 - 250 0.9958
5.3.5 Operational stability, storage stability and reusability
The stability of the IMEPP tips was studied. Several identical IMEPP tips were
prepared and tested for the digestion of denatured BSA. The IMEPP tip used for
digestion of denatured BSA after 3 months storage gave a sequence coverage of
over 80%. The IMEPP tip was also tested for reusability after digestion of the
cytochrome c spiked in plasma. A sequence coverage of over 80% could be
achieved. Figure 5-18 shows the SEM images of the morphology of the monolith
after the photografting of reactive functional monomer, enzyme immobilization
and protein digestion. A modified monolith with the desired functional groups
was successfully prepared without altering the basic morphology of the monolith.
However, the proteolytic activity of the IMEPP tip decreased after digestion of
large proteins spiked in rat plasma. This might be due to non-specific protein
adsorption which blocked the active sites on the monolith pores resulting in a loss
of activity. Nevertheless, it is important to note that the reusability of pipette tips is
not crucial in the present case since they were designed to be disposable. In
addition, the reusability of tips may not be desirable in biological applications
where it is important to avoid any possible chances of carry-over.
Chapter 5
162
(a) (b)
Figure 5-18: SEM images of porous polymer monoliths inside a PP tip after (a) VAL photografting, enzyme
immobilization and used for protein digestion and (b) the magnified part.
Chapter 5
163
5.4 Conclusions
In this chapter, a miniaturized platform enzymatic reactor for application in
bioanalysis is presented, based on a pipette tip format. The IMEPP tip showed high
hydrolytic activity and long term stability for the digestion of low- and high-
molecular weight proteins. Sequence coverages obtained with the IMEPP tip in 20
pipetting cycles, which is equivalent to a short total digestion time of 6 min, was
comparable to those yielded by the conventional in-solution tryptic digestion for 24
h. Furthermore, the IMEPP tip was also successfully applied to the digestion of
proteins spiked in rat plasma, demonstrating its potential to be used in proteomic
studies, as an alternative to commercially available tryptic digest tips. High plasma
loading capacity and good calibration curve linearity of the tips were also
demonstrated, thus indicating a wide dynamic range for quantitative analysis.
These evaluations were conducted in an industrial bioanalytical laboratory (Pfizer
Global Research and Development, Sandwich, UK) and demonstrated the
possibilities of high-throughput sample preparation for pharmaceutical and
pharmacokinetic studies, leading to faster and safer discovery of new drugs to treat
diseases. The developed IMEPP tip is highly suited for MS-based proteomic and
peptidomic analyses, for which the amount of sample is often limited.
5.5 References
[1] J. Krenkova, Z. Bilkova, F. Foret, J. Sep. Sci. 28 (2005) 1675.
[2] D.S. Peterson, T. Rohr, F. Svec, J.M.J. Frechet, Anal. Chem. 74 (2002) 4081.
[3] E. Calleri, C. Temporini, E. Perani, A. De Palma, D. Lubda, G. Mellerio, A.
Sala, M. Galliano, G. Caccialanza, G. Massolini, J. Proteome Res. 4 (2005)
481.
Chapter 5
164
[4] S. Ota, S. Miyazaki, H. Matsuoka, K. Morisato, Y. Shintani, K. Nakanishi, J.
Biochem. Biophys. Methods 70 (2007) 57.
[5] J. Spross, A. Sinz, Anal. Chem. 82 (2010) 1434.
[6] S. Wang, Z. Chen, P. Yang, G. Chen, Proteomics 8 (2008) 1785.
[7] M. Ethier, W. Hou, H.S. Duewel, D. Figeys, J. Proteome Res. 5 (2006) 2754.
[8] Z. Altun, C. Skoglund, M. Abdel-Rehim, J. Chromatogr. A 1217 (2010) 2581.
[9] M. Abdel-Rehim, C. Persson, Z. Altun, L. Blomberg, J. Chromatogr. A 1196-
1197 (2008) 23.
[10] J. Krenkova, N.A. Lacher, F. Svec, J. Chromatogr. A 1216 (2009) 3252.
[11] J. Krenkova, N.A. Lacher, F. Svec, Anal. Chem. 81 (2009) 2004.
[12] W.K. Russell, Z.Y. Park, D.H. Russell, Anal. Chem. 73 (2001) 2682.
[13] D.S. Peterson, T. Rohr, F. Svec, J.M.J. Frechet, Anal. Chem. 75 (2003) 5328.
[14] W.S. Gordon, C.S. David, Rapid Commun. Mass Spectrom. 17 (2003) 1044.
[15] D. Chelius, G. Xiao, A.C. Nichols, A. Vizel, B. He, T.M. Dillon, D.S. Rehder,
G.D. Pipes, E. Kraft, A. Oroska, M.J. Treuheit, P.V. Bondarenko, J. Pharm.
Biomed. Anal. 47 (2008) 285.
[16] Y. Li, M.L. Lee, J. Sep. Sci. 32 (2009) 3369.
[17] C. Barton, P. Beck, R. Kay, P. Teale, J. Roberts, Proteomics 9 (2009) 3058.
[18] S. Miyazaki, K. Morisato, N. Ishizuka, H. Minakuchi, Y. Shintani, M.
Furuno, K. Nakanishi, J. Chromatogr. A 1043 (2004) 19.
Chapter 6
165
C h a p t e r 6
Optimization of Permeability and Format of
IMEPP Tips for Proteomic Analysis
6.1 Introduction
In the pharmaceutical industry, the availability of highly developed tools for
analytical procedures such as separation and detection instrumentation has led to
an increase in the number of new drugs. With the advancement in analytical
instrumentation, sample preparation has become a critical component in the
analytical procedure but at the same time it has also become a bottleneck which
limits high-throughput analysis. Because of this, much effort has been devoted to
developing high-throughput sample preparation methods for toxicological and
pharmacokinetic studies. An ideal sample preparation method should be easy to
learn, environmental friendly, economical, and involve minimal manual handling
steps. Furthermore, semi- or fully-automated analytical techniques are required to
cope with the growing number of samples to be analyzed in the pharmaceutical
industry [1]. Recent developments in sample handling techniques are directed
towards miniaturization, automation and on-line coupling of sample preparation
units and detection systems to speed up the whole analytical process. The more
rapidly these measurements can be done, the faster is the progress of the drug
toward regulatory approval and the more economically viable the drug becomes
for the consumers [2].
Tip-based technology has become increasingly popular in the development
of high-throughput enzymatic digestion method for proteomic studies [3]. This
miniaturized bioanalytical format is based on the stationary phase fixed inside a
Chapter 6
166
pipette tip. The sample preparation is achieved by repeated aspirating and
dispensing cycles using a manual micropipettor [4]. A 96-tip liquid handling
robotic device has been introduced which allows 96 samples to be prepared
simultaneously in only several minutes [5]. Compared with conventional liquid
phase digestion, the tip-based digestion is easier, faster, and less expensive.
There are several commercially available enzyme tips, including DigesTip®
Trypsin from ProteoGen Bio (Pisa, Toscana, Italy) and MonoTip® Trypsin from GL
Sciences (Tokyo, Japan). More recently, a novel miniaturized SPE device, called
microextraction by packed sorbent (MEPS) was introduced by Abdel-Rehim [6].
This MEPS device was demonstrated to be compatible with the majority of the
commercially available autosamplers, as it allows sample purification,
concentration and direct injection into GC or LC systems without modification of
the chromatograph. Furthermore, this technique can be easily interfaced to GC-
and LC-MS to provide a completely automated MEPS/LC-MS or MEPS/GC-MS
system. The principles and application details of MEPS have been documented in a
review by Abdel-Rehim [6]. This technique could be of interest in pharmaceutical
and pharmacokinetic studies if the MEPS sorbent bed is immobilized with enzyme
and integrated into a liquid handling syringe which allows protein digestion,
sample clean-up and sample injection in a single step.
Since the introduction of monolithic stationary phases in the 1990s by Svec
and Tanaka, monolithic materials have been employed in enzyme immobilization
to achieve fast digestion steps from several hours to a few minutes or even seconds
[7,8]. At present, a wide variety of monomers are used for the preparation of
polymeric monolithic stationary phases. The diversity of monomers provides the
possibility of synthesizing customized polymer monolith with a desired porosity,
selectivity, pore diameter and functionality, according to the needs of specific
applications [9]. In the previous chapters, butyl methacrylate monoliths have been
Chapter 6
167
prepared in situ in 75 µm i.d. fused-silica capillaries and used for the HPLC
separation of -blockers and acidic compounds. The impact of different
polymerization parameters (monomer content, porogenic solvent and cross-linker)
on the porous properties, and hence on the separation selectivity for some acidic
drugs and -blockers, was studied. Recent developments and applications of
monolith-based immobilized enzyme reactors for proteome analysis have been
summarized in a review by Ma et al. [10].
This chapter describes the effects of the chaotropic agents, urea and
guanidine hydrochloride (GdnHCl), disulfide bond reducing reagents
dithiothreitol (DTT) and Tris(2-carboxyethyl)phosphine hydrochloride (TCEP), as
well as different buffer solutions containing calcium chloride in the protein-
containing solution used for digestion. The digestion efficiencies were evaluated
using a Mascot database search of peptide fragment fingerprint (PFF) analysis. In
addition, the IMEPP tip was prepared in different formats including an empty
monotip and a specialized glass tube. Potentially, higher sample loading capacity
and digestion efficiencies could be achieved. The influences of different polymer
monolith formulations prepared in situ in PP tips on the permeability and
digestion efficiencies were also investigated in detail.
6.2 Experimental
The general experimental details are described in Chapter 2. Detailed conditions
are elaborated in each of the figure captions.
6.2.1 Comparison of protein digestion conditions
A comparative study of protein digestion in different buffer solution (ammonium
acetate and Tris-HCl) with and without acetonitrile and calcium chloride was
performed with 0.1 mg/mL cytochrome c solution. The effects of the chaotropic
Chapter 6
168
agents, urea and guanidine hydrochloride (GdnHCl), disulfide bond reducing
reagents DTT and TCEP were evaluated with the digestion of 0.5 mg/mL BSA
solution. The composition of the solutions and reagents for the digestion are listed
in Table 6.1. The sample preparation details are summarized in Sections 5.2.1 and
5.2.2. After the digestion of proteins using soluble trypsin and IMEPP tips, the
digests were collected and analyzed via a LC-MS/MS method.
6.2.2 Protein identification using public protein sequence database
Peptide fragment fingerprints (PMFs) LC-MS/MS analyses for protein
identification were performed with the Bruker Compass DataAnalysis 4.0 software
using Mascot software (Matrix Science Ltd., UK, http://www.matrixscience.com
website) and the public database SwissProt. Protein identification based on LC-ESI-
MS/MS experiments was achieved by conversion of raw data to mgf-files. The mgf-
files were searched using Mascot against SwissProt database with mammalia
taxonomy. For proteins that were not chemically treated, such as cytochrome c,
trypsin was selected as the digestion enzyme and 3 missed cleavage sites were
allowed (precursor mass tolerance of 0.02 Da, fragment mass tolerance of 0.05 Da).
The deaminated, Gln pyro-Glu (N-term Q) and methionine oxidation were
entered as variable modifications. Peptide charge was set to 1+. All peptide mass
values were monoisotopic. Data format was set to Mascot generic and the
instrument used was ESI-QUAD-TOF. A decoy setting was selected before the
database search began. For proteins such as BSA that have already been treated
with denaturants, all the parameters were the same except that carbamidomethyl
cysteine was entered as a fixed modification and ammonia-loss, deaminated, Gln
pyro-Glu (N-term Q) and methionine oxidation were entered as variable
modifications.
Chapter 6
169
Table 6.1: Buffers and denaturants used for protein digestion.
Protein buffer In Solution IMEPP tip
50 mM ammonium acetate + 20% ACN x
50 mM ammonium acetate + 10 mM calcium chloride + 20% ACN x
50 mM TRIS-HCl + 20% ACN x
50 mM TRIS-HCl + 10 mM calcium chloride + 20% ACN x
100 mM TRIS-HCl + 10 mM calcium chloride + 20% ACN x
Denaturant
Urea + DTT + IAA x x
GdnHCl + TCEP + IAA x x
Cytochrome c
BSA
Chapter 6
170
6.2.3 Preparation of highly permeable monoliths
Surface modification in the pipette tip was done using single-step photografting
according to the procedure described in Chapter 2. Nine different compositions of
monoliths were prepared in situ in polypropylene pipette tips and all the
compositions investigated are listed in Table 6.2.
6.2.4 Manipulations of polymer monoliths in situ in polypropylene
pipette tips
Several types of IMEPP tips have been proposed, as shown in Figure 6-2. Tips (a) to
(e) are Finntip-200 extended pipette tips and (f) is a Finntip-1000 purchased from
Thermo Scientific. The tip (g) is called MonoTip empty tip 200 µL, purchased from
GL Sciences (Tokyo, Japan). A piece of capillary tubing was inserted into the filled
tip as a template to create a main channel through the immobilized bed. After
polymerization, it was removed to increase the permeability of the solid support.
There were two models of through-channel pipette tips prepared, as shown in
Figure 6-3. Both models of through-channel pipette tips were filled with 40 µL of
polymerization mixture and exposed under UV.
6.3 Results and discussion
6.3.1 Optimization of protein digestion conditions
Cytochrome c is a small protein (11.7 kDa) which is frequently used for evaluating
enzymatic digestion efficiencies. Cytochrome c contains 105 amino acids and 21
tryptic cleavage sites (i.e. Arg and Lys residues). The peptides resulted from the
digestion can be easily identified using either direct infusion or LC-MS methods. In
Chapter 4, it was described how cytochrome c was used to evaluate the optimum
amount of VAL needed for immobilization and the effects of trypsin concentration
Chapter 6
171
Table 6.2: Compositions of the polymerization mixtures (%, w/w) used for the preparation of polymer monoliths in situ
in pipette tips.
Polymerization mixtures
Monomers / Initiator / Porogens (wt%) S2M
S2M
70%
S2M
75%
S1
70%
S1
75%
Tetraglycol
thermal
Tetraglycol
UV LP1 LPM1
Butyl methacrylate 16 12 10 18 15 19 19 15 10
Ethylene glycol dimethacrylate 24 18 15 12 10
10 15
Tetra(ethylene glycol diacrylate)
21 21
Vinylazlactone
1-Propanol 42 49 52.5 42 45 40 40
1,4-Butanediol 12 14 15 21 22.5 20 20
1-decanol
Water 6 7 7.5 7 7.5
Methanol
52.5 52.5
Hexane
22.5 22.5
2,2-dimethoxy-2-phenylacetophenone 1 1 1 1 1
1 1 1
Azobisisobutyronitrile
1
Median pore diameter, nm 1297 1514 1901 1231 1499 N/A N/A
N/A N/A
Porosity, % 62 72 77 70 78 N/A N/A
N/A N/A
Chapter 6
172
Figure 6-2: Several types of IMEPP tips were prepared. (a) Tip filled with 20 µL
of polymerization mixture; (b) the tip filled with 20 µL of polymerization
mixture and exposed under UV horizontally for 2.5 to 6 min and vertically from
5 to 8 min; (c) the tip filled with 8 µL of polymerization mixture; (d) tip filled
with 20 µL of polymerization mixture, and a larger “converter” was attached to it
to allow the use of larger volume autopipette in order to give higher pipetting
pressure; (e) top part of the tip filled with 20 µL of polymerization mixture (f) 1
mL-tip filled with 20 µL of polymerization mixture; (g) MonoTip empty tip 200
µL. All the polymerizations were conducted with the sharp end of the tip
pointing downwards for 40 min and then with the sharp end up for 25 min,
unless otherwise stated.
Chapter 6
173
Figure 6-3: Two models of through-channel IMEPP tips: (a) monolithic bed in
the middle section of the tip, (b) monolithic bed at the end of the tip. The tips
were exposed horizontally under UV for 30 min.
Chapter 6
174
on immobilization. It was also mentioned in Chapter 5 that cytochrome c was used
to establish several tests in an industrial bioanalytical lab (Pfizer, UK) including
plasma loading capacity, optimum volume for digestion, time course assay, sample
clean-up and enrichment assay, and finally the qualitative and quantitative
bioanalysis.
There were two different protein digestion conditions that were used for
qualitative and quantitative analysis in Chapters 4 and 5. The reagents used in the
Pfizer Analytical Research Center (PARC, University of Tasmania, Australia) were
described in the experimental work by Krenkova et al. [7]. The standard buffer
used to make protein solution is 50 mM ammonium acetate at pH 8.75 containing
20% acetonitrile. However, a different standard buffer was used in the Pfizer
Bioanalytical Laboratory (Sandwich, United Kingdom). A 100 mM Tris-HCl buffer
was used to prepare protein solution for digestion. Calcium chloride was also
commonly added to the buffer solution because it has been reported that the
calcium chloride can provide increased resistance to changes in conditions, such as
pH or temperature [11]. The functions of calcium in stabilizing the enzyme
conformation, reducing activation energy and maintaining the molecular folding of
the denatured enzyme have also been reported [12]. As higher sequence coverage
is normally achieved in the presence of an optimum amount of acetonitrile,
acetonitrile was also added to all the protein solutions.
The enzyme activity assays for different buffers were conducted in solution,
and the assumption made that the data should be similar to that generated from
the immobilized enzyme in pipette tips. The sequence coverage of in-solution
digestion of cytochrome c using different buffers for the preparation of protein
solutions are summarized in Table 6.3. There was no significant difference in the
sequence coverage when different buffers were used. However, the ammonium
Chapter 6
175
acetate buffer gave slightly higher sequence coverage in most cases than when
Tris-HCl buffer was used. The addition of calcium chloride to the digestion buffer
did not lead to any significant increase in enzyme activity (Table 6.3); instead, it
resulted in the undesirable formation of a high salt peak on the chromatogram,
which might cause ion suppression in the MS analysis and interfere with data
analysis [Figure 6-4(b)]. Further, the high salt content of the Tris-HCl buffer led to a
reduction in the intensity of most of the peptide peaks and as a result less peptides
were identified [Figures 6-4 (a, c, d)]. It was interesting to discover that the use of
Tris-HCl buffer resulted in much higher intensity of some of the peptide peaks
(Figure 6-5) for the cytochrome c digest. This could be attributed to the fact that the
Tris-HCl buffer could create some specific cleavage sites for trypsin. Therefore, the
use of ammonium acetate buffer was preferred because it did not require desalting
steps.
For an optimum digestion of large and folded proteins, specific treatment
prior to digestion was necessary to reduce the disulfide bonds. These proteins have
to be treated by denaturing agents, such as urea or GdnHCl. Most enzymes used
for proteomic studies have shown low tolerance against denaturants. However,
immobilized enzyme has been shown to be more stable in the presence of a high
concentration of denaturing agents [7]. In addition, the disulfide bridge was
reduced using DTT or TCEP and the thiol group subsequently alkylated with
iodoacetamide (IAA) to enhance exposure of sites of the protein previously
inaccessible for proteolysis. Therefore, a comparative study was established to test
the digestion efficiencies of various combinations of denaturing, reducing and
alkylating agents.
The results of digestion of BSA by soluble trypsin and the IMEPP tip are
shown in Table 6.3. The sample pre-treated with a combination of urea, DTT and
Chapter 6
176
Table 6.3: Results of the digestion of cytochrome c and BSA using different buffers and denaturants.
In Solution IMEPP tip
buffer
50 mM ammonium acetate + 20% ACN 76
50 mM ammonium acetate + 10 mM calcium chloride + 20% ACN 75
50 mM TRIS-HCl + 20% ACN 70
50 mM TRIS-HCl + 10 mM calcium chloride + 20% CAN 70
100 mM TRIS-HCl + 10 mM calcium chloride + 20% ACN 68
Denaturant
Urea + DTT + IAA 75 66
GdnHCl + TCEP + IAA 71 52
Sequence coverage (%)
BSA
Cytochrome c
Chapter 6
177
217.1
317
245.1
254
403.7
365
422.7
107
454.2
715
473.2
454
493.6
105
536.3
104
548.9
608
584.8
160
603.7
906
614.6
471
648.8
650
667.8
401
678.3
815
694.6
848
707.3
355
739.9
184
779.4
515
803.9
674
817.8
141
856.9
212
875.8
966
907.5
483
921.4
724
939.9
382
957.1
781
977.0
001
1041.5
276
1060.5
006
1082.5
438
1105.5
759
1125.5
124
1137.5
934
1168.6
289
+MS, 0.7-1.0min #(40-62)
0.0
0.5
1.0
1.5
4x10
Intens.
200 400 600 800 1000 1200 m/z
172.9
397
218.9
018
242.9
438
260.9
549
278.9
654
292.9
814
366.8
580
390.9
027
414.9
490
432.9
609
454.2
757
473.2
495
548.9
218 572.9
497
584.8
150
603.7
965
636.7
549
648.8
684
667.8
436
682.8
482
707.3
441
750.6
849
763.3
361
775.9
843
788.6
390
+MS, 1.1-1.4min #(67-85)
0
2000
4000
6000
8000
Intens.
200 400 600 800 1000 1200 m/z
122.0
754
160.0
242
178.0
360
196.0
011
243.1
518
261.9
870
282.0
998
297.9
628
317.0
740
355.0
216
390.9
986
428.9
740
454.2
759
483.9
263
502.9
039
520.8
945
538.8
797
557.8
615
584.8
173
599.4
185
623.9
858
637.3
811
659.9
567
715.9
080
735.8
725
754.8
436
771.8
583
817.3
266
836.2
994
892.9
359
947.8
869
976.0
619
1002.8
367
1014.0
130
1059.7
899
1175.2
791
+MS, 0.6-0.9min #(35-53)
0.0
0.2
0.4
0.6
0.8
1.0
1.2
4x10
Intens.
200 400 600 800 1000 1200 m/z
122.0
753
160.0
236
178.0
355
206.0
303
243.1
518
282.0
994
317.0
737
355.0
203
390.9
981
409.1
994
426.9
767
520.7
236
544.4
715
584.8
193
603.7
955
623.9
912
659.9
585
707.1
151
738.4
876
754.8
335
781.0
415
805.2
838
819.3
846
836.3
004
855.7
719
892.9
378
938.0
947
976.0
661
1014.0
193
1049.9
944
1133.1
226
1207.0
471
+MS, 1.2-1.4min #(74-85)
0.0
0.5
1.0
1.5
4x10
Intens.
200 400 600 800 1000 1200 m/z
Figure 6-4: Direct infusion mass spectra of cytochrome c digested in different buffer solutions. (a) 50 mM ammonium
acetate + 20% acetonitrile, (b) 50 mM ammonium acetate + 10 mM calcium chloride + 20% acetonitrile, (c) 50 mM Tris-
HCl + 20% acetonitrile and (d) 100 mM Tris-HCl+ 10 mM calcium chloride + 20% acetonitrile.
(a) (b)
(c) (d)
Chapter 6
178
50 mM Tris-HCl +20% acetonitrile 50 mM ammonium acetate +20% acetonitrile
0
1
2
3
4
5x10
Intens.
0 2 4 6 8 10 Time [min] 0
1
2
3
4
5x10
Intens.
0 2 4 6 8 10 Time [min]
585.1
0
1
2
3
4
5x10
Intens.
400 500 600 700 800 m/z
585.1
0
1
2
3
4
5x10
Intens.
400 500 600 700 800 m/z
0.0
0.5
1.0
1.5
4x10
Intens.
400 500 600 700 800 m/z
Y9+1
379.2 Y8+1
492.3
Y6+1
686.4
Y5+1
799.5
Y7+1
549.3
Y4+1
913.5
0.0
0.5
1.0
1.5
4x10
Intens.
400 500 600 700 800 m/z
Y9+1
379.2 Y8+1
492.3
Y6+1
686.4
Y5+1
799.5
Y7+1
549.3
Y4+1
913.5
(a) (b)
Figure 6-5: Cytochrome c (0.1 mg/mL) after in-solution digestion in buffers (a) 50
mM Tris-HCl +20% acetonitrile and (b) 50 mM ammonium acetate +20%
acetonitrile. Top: base peak chromatograms. Middle: mass spectrometry (MS)
survey scans. Bottom: tandem mass spectra (MS/MS) of the precursor ion at m/z
585.1 corresponding to peptide TGPNLHGLFGRK, amino acids 29-39.
BPC BPC
MS/MS MS/MS
Chapter 6
179
IAA yielded slightly higher sequence coverages for both in-solution digestion and
the IMEPP tip.
The present results are in agreement with the findings of Spross and Sinz
who reported that urea was a better denaturant than GdnHCl when DTT was used
as the reducing agent and IAA was used as the alkylating agent [9]. Although the
use of GdnHCl as denaturant and TCEP as reducing agent resulted in a lower
sequence coverage, it was found that the LC-chromatograms of the digestion gave
a cleaner separation profile and resulted in higher intensities of some of the peptide
peaks (Figure 6-6). Therefore, the combination of urea, DTT and IAA was probably
better suited for qualitative analysis whereas GdnHCl, TCEP and IAA could be
used for quantitative analysis of proteins.
6.3.2 Development of a highly permeable IMEPP tip
Despite the high loadability of the IMEPP tips for the protein digestion in rat
plasma of up to 40 µL, they were limited by high backpressure caused by the large
amount of sorbent used. Most of the pipetting tasks have to be performed by
attaching the IMEPP tips to a curved syringe, as shown in Figure 2-4, to give a
higher pipetting pressure. Therefore, different formulations of monolith and
physical formats of the polymer monoliths in situ in polypropylene pipette tips
were prepared in terms of the positions and the amount of polymerization mixture
used, in order to give the monolith support to a higher permeability.
6.3.2.1 Preparation of highly permeable monoliths
There are many parameters that influence the porous properties of monoliths,
including the type and concentration of the porogenic solvent(s), the
polymerization temperature and time, and the percentage of cross-linker and
initiator. Based on the preliminary experiments reported in chapter 3, monomer
mixtures consisting of BMA and EDMA, and a porogenic solvent mixture
Chapter 6
180
Figure 6-6: Separation of peptides resulting from BSA digestion (0.5 mg/mL) using the IMEPP tip and soluble enzyme by
the combining different denaturants and reducing agents. Other conditions are the same as in Figure 5-4.
Chapter 6
181
containing 1-propanol and 1,4-butanediol, were used for the preparation of the
IMEPP tips. Monolith formulations with the monomer Tetra(ethylene glycol)
diacrylate were also investigated because they had been prepared in a capillary
and reported with large pore sizes (~7 µm). Yu et al. reported that the
polymerization mixture containing BMA and EDMA with a binary porogenic
solvent of hexane and methanol yielded a monolith with large pore sizes (~8 µm)
[13]. The porogenic solvent is the most important parameter for controlling the
porous structure. Therefore, the ratio of monomer mixture to porogenic solvent in
the polymerization mixture was slightly modified in order to increase the extent of
through-pores and to facilitate low backpressure during enzyme digestion in the
IMEPP tip. Compositions of the polymerization mixture are shown in Table 6.2.
The monolith S2M, HEMA, tetraglycol thermal and tetraglycol UV were prepared
from a polymerization mixture containing 40% (w/w) of the monomer mixture and
60% (w/w) of porogenic solvent. To improve the through-pores, the composition of
porogenic solvent in the polymerization mixture was increased from 60% (w/w) to
70% (w/w) in S2M 70% and S1 70%, and increased from 60% (w/w) to 75% (w/w) in
S2M 75% and S1 75%, respectively. The pore diameters and total porosities of the
prepared monoliths were investigated through mercury porosimetry.
From Table 6.2, it can be observed that the composition of had an influence
on the porous properties and morphologies of the monolithic support. The pore
diameters of the monoliths S2M, S2M 70% and S2M 75% increased slightly as the
proportion of porogenic solvent in the polymerization mixture increased. In most
cases, the total porosities also increased as the proportion of porogenic solvent
increased (Table 6.2). Hence, increasing the porogen-to-monomer ratio is a
straightforward method to increase the pore size and porosities, and consequently,
to increase permeability during operations. However, this approach may also
Chapter 6
182
decrease the rigidity of the macroporous polymer [14]. Figure 6-7 displays SEM
images of the monolithic structures of the monoliths formed in the tips. Although
the UV-initiated polymerization is usually performed within a short period
compared to thermal initiation, the SEM images clearly showed the uniform
porous structure across the monolithic support of the pipette tip. The difference in
the pore diameters of S1 70% compared to S2M was not significant. The S1 75%
and S2M 75% monoliths prepared within the pipette tips were very porous, but
both monoliths collapsed after being washed with acetone. In addition, four of the
monoliths studied, including tetraglycol thermal, tetraglycol UV, LP1 and LPM1
formed an emulsion in the tip. Therefore, they were not further studied. The
permeabilities of S2M and S2M 70% were further investigated by preparation in
polypropylene pipette tips.
6.3.2.2 Manipulation of IMEPP tip formats
Different formats of macroscopic monoliths are commonly prepared, such as disks,
rods (or columns) and polypropylene tips. They are often commercially available
and produced by several companies such as BIA separations (Ljubljana, Slovenia)
under the trade name CIM® Disk, or as separation columns (ProSwiftTM, Dionex
Corporation, USA). Monoliths in microscopic formats, such as capillary columns,
silica steel tubing and microfluidic chips have also been widely developed [14].
Based on the preliminary experiments studied in the pipette tip format,
further adjustments were made in order to achieve more permeable tips that were
compatible with autopipettes. Figure 6-8 shows different models of pipette tips
prepared with the S2M monolith and Table 6.4 summarizes the physical properties
of these tips. Tip model (b) was prepared by using a shorter UV exposure period.
A 2.5 min exposure was not sufficient for the polymerization to occur.
Consequently, the monolithic support collapsed when it was washed with acetone
Chapter 6
183
S2M 60% S2M 70% S2M 75%
S1 70% S1 75%
Figure 6-7: SEM images of porous polymer monoliths inside the PP tips.
Chapter 6
184
after polymerization. UV initiation for 3 min resulted in partial polymerization of
the polymerization mixture, leaving a large void between pores and therefore the
tip was only partially compatible with the autopipette. As soon as the
polymerization exceeded 4 min, the monoliths were completely polymerized and
required higher pipetting pressure (Table 6.4).
The IMEPP tip that was prepared by polymerization using 8.5 min exposure
was fully compatible with the autopipette (Table 6.4). Close examination of SEM
images revealed that the polymer monolith was not completely attached to the tip
wall. However, the polymer monolith was physically stable in the pipette during
use (Figure 6-9). Meanwhile, the polymer monolith prepared in models (c, d, e, f)
allowed only partial compatibility with autopipettes due to the limited
backpressure experienced for suction operations (Table 6.4). The preparation of
IMEPP tips in the different formats did not compromise the immobilized enzyme
activities. At least 74% sequence coverage was achieved for the digestion of
cytochrome c (0.1 mg/mL) using these different IMEPP tip formats.
Tip model (e) was initially intended to simulate a commercially available
enzyme tip, namely MonoTip® Trypsin. This tip model yielded a larger internal
i.d. of about 5 mm. A shorter monolith support prepared in the pipette tips could
give higher permeability (Figure 6-10). The monoliths S2M and S2M 70% were
further prepared in this tip model. The tip with monolith S2M only showed partial
compatibility with the autopipette, similar to that prepared in the Finntip-200
format. A highly permeable tip was achieved by filling 15 µL of S2M 70% into a
MonoTip and polymerizing under UV for 20 min. This monolith provided a
permeable through-pore size which could hold the polymer monolith in place
while the solution was aspirated or expelled in and out using autopipette.
Although this may also be achieved using 10 µL of S2M mixture in the MonoTip, a
Chapter 6
185
Figure 6-8: Several models of IMEPP tips were prepared. The labels (a) to (f) are
indicated in Figure 6-2, (g) through-channel IMEPP tips.
(a) (b) (c) (d) (e) (f) (g)
Chapter 6
186
Table 6.4: Physical properties of monoliths prepared in different formats.
* Partial compatibility meant that the solution could be aspirated but not expelled in and out of the tips or vice versa.
Tips
model
Amount of
polymerization
mixture (µL)
Polymerization
position
Polymerizatio
n time (min) Stationary phase
Compatibility
with autopipette
(200uL)
1 (a) 20 vertical 30 ok no
2 (b) 20 horizontal 2.5 collapsed NA
3 (b) 20 horizontal 3 ok partial
4 (b) 20 horizontal 4 ok no
5 (b) 20 horizontal 5 ok no
7 (b) 20 horizontal 6 ok no
8 (b) 20 vertical 5 collapsed no
9 (b) 20 vertical 6 collapsed no
10 (b) 20 vertical 7 collapsed no
11 (b) 20 vertical 8.5 ok yes
12 (c) 5 vertical 30 ok Partial
13 (c) 7 vertical 30 ok no
14 (d) 20 vertical 30 ok Partial
15 (e) 20 vertical 30 ok Partial
16 (f) 20 vertical 30 ok Partial
Chapter 6
187
Figure 6-9: SEM images of polymer monolith in pipette tip after the tip was
exposed vertically for 8.5 min under UV.
Figure 6-10: Photo of (a) MonoTip empty tip and (b) MonoTip with polymer
monolith.
(a) (b)
Chapter 6
188
larger monolith support in this case would be advantageous for enzyme
immobilization. Sequence coverage of 80% was obtained using this S2M 70%
MonoTip for the digestion of cytochrome c.
A main channel was created through the immobilized bed by inserting a
piece of capillary tubing into a tip filled with polymerization mixture and then
removed after polymerization. This method of creating a flow channel in the
monolith was first demonstrated by Hsu et al. to fabricate disposable plastic
microtips, namely EasyTip, for SPE as an off-line sample preparation of biological
samples [15]. Most importantly, the through-channel created within the
immobilized bed did not affect the functionality of the EasyTip. The recovery
percentage of the tryptic digest samples loaded onto the EasyTip was nearly 100%
[15]. The same technique was in applied to the IMEPP tips. Up to 40 µL (~6 mg) of
polymerization mixture was used in the through-channel IMEPP tips to increase
the loadability since this system was not restricted by the pipetting backpressure
[Figure 6-8 (g)]. Therefore, using this through-channel technique, the permeability
problem was overcome easily. Figure 6-11 shows a SEM photograph of the
monolithic structure with a through-channel. The monolith was attached to the tip
wall and no cracks were observed.
The monolithic support in the through-channel IMEPP tips was
immobilized with trypsin and the digestion efficiencies were assessed using
standard procedures. Figure 6-12 demonstrates the excellent digestion efficiency of
the through-channel IMEPP tips since no significant difference in sequence
coverage was observed compared to in-solution digestion as well as the IMEPP tip
without through-channel (Table 6.5). However, additional peptide peaks were
observed between the in-solution and in-tip digestion (Figure 6-12). To further
confirm that these peptide peaks resulted from the digestion, two different
Chapter 6
189
Figure 6-11: Through-channel in IMEPP tip created by inserting a piece of
capillary tubing into the tip filled with polymerization mixture, and then
removed after polymerization.
Chapter 6
190
Figure 6-12: Separation of peptides resulting from cytochrome c digestion (0.1 mg/mL) using the soluble trypsin and
through-channel IMEPP tips (a) and (b). Digestion and separation conditions are the same as in Figure 4-10.
Chapter 6
191
Table 6.5: Results of the digestion of cytochrome c and BSA using soluble
trypsin, IMEPP tip with and without through-channel.
In Solution
IMEPP tip without through-channel
IMEPP tip with through-channel
Sequence coverage (%)
Cytochrome c 81 82 77
BSA 75 66 67
Chapter 6
192
surrogate peptides were extracted from the LC-MS chromatograms for
comparison. Figure 6-13 shows two different surrogate peptides with amino acid
sequences EETLMEYLENPK and TGQAPGFTYTDANK resulting from the tryptic
digest of cytochrome c using in-solution digestion and through-channel tip
digestion. The results clearly show that the intensity of surrogate peptides
improved by 20 to 30 times for the through-channel mode.
Protein identification by Mascot software showed that the additional
peptide peaks from the through-channel IMEPP tips digest were mainly trypsin
residues from the immobilization process (Figure 6-14). This could be due to the
self-digestion of immobilized trypsin which was not completely washed out and
was trapped or absorbed on the polymer monolith during immobilization. Thus,
during the pipetting process, the digested trypsin was eluted into the sample
solution, and caused the additional peptides on the LC-MS chromatograms.
Therefore, the through-channel IMEPP tips were washed carefully with water and
digestion buffer before used. Figure 6-15 shows that there were slightly fewer
peptides observed for the LC-MS chromatogram of the protein digested using
through-channel IMEPP tip after being washed with water and digestion buffer.
However, a significant amount of additional peptides remained in comparison to
the in-solution digestion. The separation by LC-MS/MS was very sensitive and
even a small amount of trypsin leaching from the polymer monolith might result in
significant additional peaks. However, even when the tip was washed with the
water and buffer, the digestion efficiency was not compromised as the enzyme was
immobilized strongly on the solid support with covalent bonds. Through-channel
IMEPP tips exhibited high activity for digestion of high proteolytic resistivity
proteins, such as BSA, with similar sequence coverage obtained using in-solution
digestion (Figure 6-16).
Chapter 6
193
(a)
(b)
Figure 6-13: Extracted ion chromatograms of surrogate peptides with (a) amino
acid sequences EETLMEYLENPK and (b) TGQAPGFTYTDANK resulting from
the tryptic digest of cytochrome c using in-solution and through-channel tips
digestion.
Chapter 6
194
As mentioned earlier, the ideal monolith morphology in pipette format
should contain larger amount of monolith plug for enzyme immobilization to
achieve higher digestion capacity while still permeable enough for the protein to
flow convectively through the interstices and interact with enzyme-immobilized
surface. In this case, the design of Figure 6-3 is preferable because this design
fulfills the requirement of allowing large amount of monolith support to be used
while maintaining high permeability because of the through-channel. Most
importantly, this design is compatible with the autopipette. The design from Figure
6-3 does not significantly enhance sequence coverage of enzymatic digestion.
However, preliminary results on showed that the intensity of surrogate peptides
improved by 20 to 30 times digested using through-channel enzyme tip, which is
extremely useful for quantitative bioanalysis (Figure 6-13). This result has to be
confirmed using MRM quantitative method.
Figure 6-14: Database searched using Mascot software against SwissProt
database after cytochrome c digest was analyzed by LC-MS/MS and exported as
mgf-files.
Chapter 6
195
Figure 6-15: Comparison of LC-MS chromatogram for the digestion of cytochrome c using through-channel IMEPP tip (a)
before and after being washed with water and digestion buffer.
Chapter 6
196
6.3.3 Preparation of immobilized enzyme polymer monolith in glass
tube for microdigestion
MEPS is an automated version of SPE which was first developed by Abdel-Rehim
in 2003. MEPS has been growing in popularity for the extraction and concentration
of a wide range of analytes in different matrices (urine, plasma and blood) ever
since it was made commercially available [6]. Most importantly, it has been used
on-line with instruments for high-throughput sample preparation. One of the
ultimate goals in this project is to develop miniaturization devices for protein
digestion which meet the demands of automated high-throughput performance.
Thus, the format of this device should be easily prepared, replaceable and easily
integrated with the analysis without further modification.
A special glass tube (~30 µL) with a needle insert was investigated as a
potential format for a high-throughput sample preparation device (Figure 6-17). In
order to anchor the monolithic structure to the glass tube wall through covalent
bonding, the glass tube was surface-modified using the same procedure described
in Section 2.3.3. Following this, the enzyme-immobilized polymer monolith (20 µL
or 3 mg) was synthesized in the glass tube using the standard procedures for
preparing polymer monolith, photografting of VAL functionality and enzyme
immobilization. The glass tube was positioned between the syringe barrel and
needle while the protein digestion took place on the immobilized bed (Figure 6-18).
To evaluate the digestion efficiency of this new format, a protein solution
containing 0.1 mg/mL of cytochrome c was used for the digestion and its
performance was compared to that of a standard in-solution digestion. Protein
solution was drawn through the immobilized bed within the glass tube and
dispensed for about 20 cycles manually using the syringe attached. The sample
was then analyzed directly with LC-MS/MS and identified by the Mascot software.
Chapter 6
197
Figure 6-16: Separation of peptides resulting from BSA digestion (0.5 mg/mL) using the through-channel IMEPP tip and
soluble enzyme.
Chapter 6
198
Figure 6-17: Photograph of a glass tube with needle insert.
Figure 6-18: Glass tube with immobilized bed and attached to a 3 mL syringe.
Blunt needle
Immobilized enzyme polymer monolith
3 mL Syringe
Chapter 6
199
Sequence coverage of 74% was obtained for a 20 cycle digestion which required
only five min for the whole digestion process.
6.4 Conclusions
In this chapter, the effects of using different buffer solutions for protein digestion
were investigated. The combination of ammonium acetate and acetonitrile yielded
better results compared to protein digestion in Tris-HCl buffer with the addition of
calcium chloride. Also, the combination of urea (up to 8 M), DTT and IAA yielded
better digestion efficiencies for the optimum digestion of large and folded proteins
without damaging the trypsin activities. Butyl methacrylate-based monoliths were
prepared using various compositions of the polymerization mixture in order to
increase the through-pores of the immobilized bed. It was shown that increasing
the porogen/monomer ratio led to increases in the pore sizes and total porosities of
monoliths, and in turn this gave higher permeability for the suction operations.
Several formats of IMEPP tips were also developed to work concurrently with an
autopipette. It was found that an IMEPP tip prepared by polymerization of 8.5 min
exposure under UV and the MonoTip prepared with S2M 70% were fully
compatible with the autopipette. Both IMEPP tip formats gave higher permeability.
An IMEPP tip with a through-channel was also used to demonstrate the potential
of compatibility with an autopipette without compromising enzyme digestion
efficiency. A new format for enzyme digestion that has been miniaturized to work
with a syringe further expands the possibility for high-throughput sample
preparation that can be performed either manually or in conjunction with an
autosampler to allow protein digestion, sample clean-up and sample injection
accomplished in a single step.
Chapter 6
200
6.5 References
[1] Z. Altun, C. Skoglund, M. Abdel-Rehim, J. Chromatogr. A 1217 (2010) 2581.
[2] Z. Altun, L.G. Blomberg, M. Abdel-Rehim, J. Liq. Chromatogr. & Rel.
Technol. 29 (2006) 1477.
[3] S. Ota, S. Miyazaki, H. Matsuoka, K. Morisato, Y. Shintani, K. Nakanishi, J.
Biochem. Biophys. Methods 70 (2007) 57.
[4] L. Xu, Z.G. Shi, Y.Q. Feng, Anal. Bioanal. Chem. (2010).
[5] Z. Altun, A. Hjelmstroem, L.G. Blomberg, M. Abdel-Rehim, J. Liq.
Chromatogr. & Rel. Technol. 31 (2008) 743
[6] M. Abdel-Rehim, J. Chromatogr. A 1217 (2010) 2569.
[7] J. Krenkova, N.A. Lacher, F. Svec, Anal. Chem. 81 (2009) 2004.
[8] J. Spross, A. Sinz, Anal. Bioanal. Chem. 395 (2009) 1583.
[9] J. Spross, A. Sinz, Anal. Chem. 82 (2010) 1434.
[10] J. Ma, L. Zhang, Z. Liang, W. Zhang, Y. Zhang, J. Sep. Sci. 30 (2007) 3050.
[11] K.R. Bandivadekar, V.V. Deshpande, Biotech. Lett. 16 (1994) 179.
[12] M. Matsubara, E. Kurimoto, S. Kojima, K.-i. Miura, T. Sakai, FEBS Lett. 342
(1994) 193.
[13] C. Yu, M. Xu, F. Svec, J.M.J. Fréchet, J. Polym. Sci. Part A: Polym. Chem. 40
(2002) 755.
[14] E.G. Vlakh, T.B. Tennikova, J. Sep. Sci. 30 (2007) 2801.
[15] J.L. Hsu, M.K. Chou, S.S. Liang, S.Y. Huang, C.J. Wu, F.K. Shi, S.H. Chen,
Electrophoresis 25 (2004) 3840.
Chapter 7
201
C h a p t e r 7
General Conclusions
The use of polymeric monolithic materials for bioanalysis has increased over the
recent years. These materials have a number of desirable properties which include:
1. Continuous structure of interlaced through-pores and skeleton that
provides high permeability to enable chromatographic separations at
extremely high flow-rates.
2. Inexpensive, requiring no frits and easily prepared in situ in different
formats by thermal-, UV- and γ-radiation.
3. Porous properties can be controlled by varying the compositions of the
polymerization mixture, tailored to suit separations of large and small
molecules.
4. Can be modified to feature almost any functionality; ion-exchange, affinity,
chiral, mixed-mode, restricted access, hydrophobic and hydrophilic, in
order to suit the stationary phases for specific analytes.
5. Biocompatibility and high stability over a wide pH range, which is crucial
when dealing with biological samples.
Firstly, the SPMA-based monoliths were prepared both in bulk and inside
Teflon® -coated UV transparent fused-silica capillaries using photoinitiated free
radical polymerization. The relationship between the porogenic solvent
composition and cross-linker with regard to morphologies, pore size distributions
and separation performance were investigated. It was shown that the pore sizes
Chapter 7
202
and separation properties of monoliths could be controlled by varying of the
porogenic solvent ratio between 1,4-butanediol and 1-propanol. Increasing the
percentage of 1-propanol relative to that of 1,4-butanediol in the polymerization
mixture diminished the pore size in the prepared SPMA monoliths. Among the
first set of columns (S1-S7) prepared using higher functional monomer/cross-linker
ratio, the best separation of a mixture of target analytes was achieved with the
porogen ratio 1,4-butanediol:1-propanol:water 30:60:10 (% w/w/w) for the S1
column. This column exhibited good repeatability for run-to-run separation but
showed non-robustness for column-to-column and batch-to-batch separations. In
the second set of columns (S1M-S3M), the proportion of cross-linker (EDMA) in
the monomer mixture was switched with that of the functional monomer (BMA),
with the compositions BMA:SPMA:EDMA 16:0.4:23.6 (% w/w/w). The S2M column
demonstrated the best separation performance with satisfactory column
permeability suitable for an acceptable level of separation of acidic drugs and β-
blockers. The S2M column exhibited minimal column-to-column and batch-to-
batch differences in the majority of the investigated chromatographic parameters.
Therefore, SPMA-based monolithic columns with a higher amount of cross-linker
(SM-monoliths) exhibited better repeatability than those polymerized with less
cross-linker (S-monoliths). Furthermore, the separation mechanism of the
investigated compounds was shown to be based on a mixed-mode functionality of
the SCX-RP monolith. The present work has demonstrated the high
permeability (calculated using Darcy’s Law) of the developed monoliths.
Although it was not thoroughly explored in this project, the developed
monoliths could potentially be used for extremely high flow-rates separations.
Thus, further studies are warranted. Moreover, as it has not been fully
demonstrated in the current work that tailored monoliths could provide
Chapter 7
203
separations of large and small molecules, future studies may focus on improving
the developed materials for the efficient separations of large and small molecules.
A novel trypsin-immobilized polypropylene pipette tip prepared with the
newly optimized porous polymer monolith was developed. Disposable pipette tips
were surface-modified before in situ synthesis of a plug of polymer monolith as an
immobilized bed. A single photografting step using a stock solution of
modification mixtures containing MMA:EDMA:BP resulted in better attachment of
the polymer monolith on the polypropylene surface. A fluorescence assay
indicated that UV light was capable of penetrating the polypropylene wall,
enabling surface grafting of reactive functionalities (VAL) for trypsin
immobilization. The concentrations of VAL in the grafting mixture as well as the
grafting time were optimized, and the optimum trypsin concentration for
immobilization was also determined. The first demonstration of proteolytic activity
for the immobilized enzyme polypropylene pipette tips was carried out using a
solution of cytochrome c. The preliminary results obtained were very promising as
very high sequence coverage was achieved within a very short digest time of only
a few minutes.
The digestion performances of the IMEPP tips were further characterized
and evaluated using MicrOTOF-Q quadrupole time-of-flight MS and a LC-MS/MS
system including an AB SCIEX Triple Quadrupole™ 5500 mass spectrometer for
the application of bioanalysis. The IMEPP tip showed a very good catalytic efficacy
for different proteolytic resistivity proteins, ranging from 2.8 kDa to high
molecular mass proteins, such as hIgG of 150 kDa. Direct comparison of the
digestions achieved with IMEPP tips and soluble trypsin clearly demonstrated the
advantages of the former as they afforded much higher digestion efficiencies
within short periods of time. Further, IMEPP tips were also successfully applied to
Chapter 7
204
biological sample preparation for qualitative and quantitative analysis in an
industrial bioanalytical laboratory (Pfizer Inc., Sandwich). The developed IMEPP
tips exhibited high plasma loading capacity of up to 40 µL of plasma, which could
be used to yield the highest digestion efficiency. A time course assay demonstrated
that the 30 min IMEPP tip digestion yielded similar results to the 24 h in-solution
digestion. Although IMEPP tips did not possess significant sample clean-up and
enrichment functionalities, nonetheless IMEPP tips eliminated the enzyme
autodigestion which would have led to ion suppression in the MS analysis.
Calibration curves were established for different target proteins and demonstrated
relatively good linearity over a wide range from 40 ng/mL to 1000 ng/mL. IMEPP
tips showed long-term stability and reproducibility. The results obtained were
superior to those of commercially available tryptic digest tips.
The author’s laboratory and the Pfizer bioanalytical laboratory used
different digestion buffers and denaturants. Therefore, experiments were
conducted to evaluate the digestion efficiencies of the two systems. Digestion
buffer comprising 50 mM ammonium acetate, pH 8.75 containing 20% acetonitrile
yielded better results compared to digestion in Tris-HCl buffer. In addition, the
combination of urea, DTT and IAA gave better digestion of reduced and alkylated
proteins. Additionally, the monolith plug in the pipette tip was varied to increase
the extent of through-pores and to facilitate the suction operation during enzyme
digestion in the IMEPP tip. A highly permeable tip was achieved by filling 15 µL of
S2M 70% in empty MonoTip® Trypsin, with polymerization under UV for 20 min.
Newly developed through-channel IMEPP tips could also be used to overcome the
permeability problem. Most importantly, these modified IMEPP tips allowed
compatibility with autopipettes without compromising digestion performance.
Trypsin-immobilized polymer monolith was also prepared within a syringe-
Chapter 7
205
compatible glass tube and the protein digestion efficiency was evaluated.
Preliminary results showed that the enzyme on the immobilized bed maintained
high proteolytic performance in this special format.
Applications of the robust and repeatable monolithic columns and IMEPP
tips developed in this work can be expected to be further expanded in the future.
Further studies to achieve this goal would include:
Monolithic columns developed were prepared by copolymerization of
BMA, EDMA and SPMA, with the sulfonate ions contributing to the cation-
exchange functionalities of the polymer chains. However, the cation-
exchange capacity was relatively low. One alternative approach is to
introduce this functionality by post-photografting the monolithic support
with a desired functional monomer. The mixed-mode monolith sorbent
produced could be further tested for separation performance (e.g. HPLC,
LC-MS) or used as the bulk material for solid-phase extraction of large and
small molecules.
The tip-shape enzyme reactor has the advantage of easy applicability. With
further development, this highly efficient enzyme reactor based on the
pipette tip format can be adopted readily in the current setup of any
laboratory for manual operation using an autopipette or by integrating it
with an automated 96-tip robotic device for high-throughput sample
preparation of bio-substances in pharmaceutical and pharmacokinetic
studies, enabling faster and safer discovery of new drugs.
So far, the IMEPP tips have only been used in the digestion of standard
proteins. These tips could be applied in a wider range of biological samples
of pharmaceutical interest.
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206
Although this project has only demonstrated the immobilization of trypsin
on polymer monoliths in pipette tips, immobilization of other enzymes (e.g.
LysC and PNGase F) using these supports will also be feasible. It is also
possible to prepare multiple enzyme reactors in a range of different formats
with different parameters such as in situ in capillary, different internal
diameter columns, microchips, syringe barrels, syringe filters and spin
columns, leading to significant commercial potential. These designs also
mean that they can be applicable to all types of bioanalytical problems.
It is also possible to prepare the immobilized bed in 96-well plates. The
lower part of each well can be removed. The proteins samples are digested
while eluting through the 96 wells and are subsequently injected for sample
analysis (Figure 7-2).
A monolith in a glass tube could be fitted into a glass syringe and connected
to an autosampler for on-line sample preparation prior to MS analysis.
Further modification of the monolith functionalities may enable analysis of
complex matrices, e.g. using two monolithic phases with different
functionalities, thus providing the possibility of extraction, enrichment,
digestion and injection in a single step for analysis (Figure 7-3).
Chapter 7
207
Figure 7-2: 96-well plate with immobilized bed.
Figure 7-3: Glass tube with two layers of monolithic phases fitted with gas tight
syringe.
Ion-exchange phase
Immobilized enzyme bed
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